Home > From juergen.ostwald at medizin.uni-rostock.de Mon Apr 3 03:57:52 2000

From juergen.ostwald at medizin.uni-rostock.de Mon Apr 3 03:57:52 2000

From juergen.ostwald at medizin.uni-rostock.de Mon Apr 3 03:57:52 2000
From: juergen.ostwald at medizin.uni-rostock.de (Juergen Ostwald)
Date: Mon, 03 Apr 2000 10:57:52 +0200
Subject: Thanks for Cell culture: Epithelial cells or fibroblasts??
Message-ID: <38E85D10.9B75D7FD@medizin.uni-rostock.de>


Dear Flowers,Thanks for all the replies concerning: Cell culture:
Epithelial cells or fibroblasts??
I've get about more than 10 answers with good and helpfull ideas.
Anybody who will participate in this question will get this material
from me-please mail me.
Thank your very much
and good success.
J?rgen Ostwald



From kingl at pilot.msu.edu Mon Apr 3 08:31:31 2000
From: kingl at pilot.msu.edu (Louis King)
Date: Mon, 03 Apr 2000 09:31:31 -0400
Subject: What experience does anyone have with Spectra Physics water
cooled lasers in FACS units?
Message-ID: <3.0.5.32.20000403093131.007e93a0@pilot.msu.edu>


To: cyto-inbox

I am compenplating replacing a Spectrum laser with a laser better
suited for UV excitation. Has anyone had any experience with Spectra
Physics current production lasers in a flow cytometry application? Has the
laser yielded dependable service in the UV line(s) with good DNA CVs? Has
Spectra Physics been able to provide prompt phone and/or field service?


Louis King, Ph.D.

Michigan State University Flow Cytometry Center
Department of Biochemistry, Rm 419
Michigan State University
East Lansing, MI 48824

Phone: 517-355-1536
FAX: 517-353-9334
E-Mail: kingL at pilot.msu.edu


From l.barber at pmci.unimelb.edu.au Sun Apr 2 17:33:10 2000
From: l.barber at pmci.unimelb.edu.au (Barber, Lesley (PMC))
Date: Mon, 03 Apr 2000 08:33:10 +1000
Subject: Mouse lymphocyte separation
Message-ID: <58983447B50ED311954300A0C98A352501497E62@PMC-MAIL01>


We use Nycoprep (mouse variant) from Nycomed
Mrs. Lesley Barber
Edith Margaret Collie Centre for Blood Cell Therapies
Div. Haematology/Medical Oncology
Peter MacCallum Cancer Institute
East Melbourne, 3002
VICTORIA , AUSTRALIA
Tel 61 3 9656 1957
Fax 61 3 9656 1811


> -----Original Message-----
> From: Cathryn Broderick [SMTP:opt074 at abdn.ac.uk]
> Sent: Friday, March 31, 2000 4:16 AM
> To: Cytometry Mailing List
> Subject: Mouse lymphocyte separation
>
>
> Does anyone know of a method for separating mouse
> lymphocytes from cell suspension, similar to human
> lymphocytes and histopaque?? That is, is there a
> commercial product available specifically for mouse
> lympocytes, or which protocols are people currently using
> instead?
>
> Thanks for any help...
>
> Cathryn
>
> ----------------------
> Cathryn Broderick
> c.broderick at abdn.ac.uk
>
>


From B_Hooibrink at CLB.nl Mon Apr 3 07:45:27 2000
From: B_Hooibrink at CLB.nl (Hooibrink, Berend)
Date: Mon, 3 Apr 2000 14:45:27 +0200
Subject: CD14/beta 7
Message-ID: <633112D354E6D111BA540008C724D26401096920@clbmx01.clb.nl>


Hi Becky,

I forwarded your problem concerning cd14 expression on eosinophils to
somebody on our lab and got the following answer back:

Becky,

In the past, we have tried to determine the expression of CD14 on isolated
human eosinophils. From the results, we interpreted that the expression of
CD14 on eosinophils is very low, if any. In addition, we did not find
effects of LPS in the presence of LBP (as you probably know, CD14 is the
receptor for the LPS/LBP complex) on isolated human eosinophils. This is
probably not the answer you were looking for, but nevertheless.

Anton Tool
Department of experimental haematology
CLB Amsterdam

Regards,
Berend Hooibrink
FACSlab
CLB Amsterdam
B_Hooibrink at CLB.NL <mailto:B_Hooibrink at CLB.NL>




-----Original Message-----
From: Becky Kelly [SMTP:Eak at medicine.wisc.edu]
Sent: dinsdag 28 maart 2000 1:09
To: Cytometry Mailing List
Subject: CD14/beta 7


Does anyone have experimental evidence for CD14 expression on
eosinophils and/or beta
7 expression on monocyte/macrophages?
Thanks, Becky




From lkrueg at ix.netcom.com Sun Apr 2 09:18:52 2000
From: lkrueg at ix.netcom.com (lori krueger)
Date: Sun, 2 Apr 2000 10:18:52 -0400
Subject: platelet fixation
References: <v01530566b50a6c8416b6@[128.143.75.8]>
Message-ID: <01c601bf9cae$60013e40$dea4fea9@8skha>


Bill,
P-Selectin expression on platelets is formaldehyde fixation stable. We
routinely use Polysciences methanol free Ultrapure 10% formalin stock
diluted in dPBS or 10mMHepes/saline. First dilute stock formalin to a 2%
solution. You can add whole blood (we routinely use 3.2% citrate
anticoagulant) 1:1 (vol:vol) to 2% formalin. Dilute fixed blood 1/10 prior
to labeling.
Good Luck,
Lori Krueger

----- Original Message -----
From: William Ross <wgr8w at virginia.edu>
To: cyto-inbox
Sent: Friday, March 31, 2000 10:56 AM
Subject: platelet fixation


>
> does anyone no of a good platelet fixation procedure for the study of
> selectin expression? It is a study of myocardial infarction patients and
> timing the heart attacks is difficult and we're trying to fix the purified
> prep for later analysis.
>
> thanks in advance.
>
> Bill Ross
>
> William (Bill) Ross
> Director - FACS Core Facility
> MR-4, Box G005
> University of Virginia School of Medicine
> Charlottesville, VA 22908
> office (804) 982-1586
> fax (804) 924-1221
> e-mail:wgr8w at virginia.edu
>
>



From craig.turner at nbs.nhs.uk Mon Apr 3 03:55:41 2000
From: craig.turner at nbs.nhs.uk (craig.turner@nbs.nhs.uk)
Date: Mon, 3 Apr 2000 9:55:41 +0100
Subject: platelet fixation
Message-ID: <TFSFHQLC@nbs.nhs.uk>


So far I haven't used a fixative for platelets that doesn't increase the base line for
P-selectin expression. However, for studying relative levels, then fixation with 1%
PFA for 5 mins at room temp. This will allow you to read the samples the next day. I
would suggest you analyse the samples in citrated blood asap. Mody et al (1999) have
found that using CTAD anticoagulated samples provide a longer period for analysis
without in vitro activation. Of course reading Alan Michelson's reviews on analysis
of platelet activation by flow cytometry will help you decide.

Craig Turner

----------
From: wgr8w at virginia.edu
To: cyto-inbox
Subject: platelet fixation
Date: Friday, March 31, 00 20:50


does anyone no of a good platelet fixation procedure for the study of
selectin expression? It is a study of myocardial infarction patients and
timing the heart attacks is difficult and we're trying to fix the purified
prep for later analysis.

thanks in advance.

Bill Ross

William (Bill) Ross
Director - FACS Core Facility
MR-4, Box G005
University of Virginia School of Medicine
Charlottesville, VA 22908
office (804) 982-1586
fax (804) 924-1221
e-mail:wgr8w at virginia.edu




From rin0dss at bumed30.med.navy.mil Mon Apr 3 09:55:31 2000
From: rin0dss at bumed30.med.navy.mil (Douglas S. Smoot)
Date: Mon, 3 Apr 2000 10:55:31 -0400
Subject: sheath fluid for Calibur
Message-ID: <1.5.4.16.20000403105530.533719aa@bumed30.med.navy.mil>


We routinely use filtered DeIonized water in our FACScan and Elite flow
cytometers. The exception, of course, is when we sort. In the past when we
were running Indo-1 Calcium measurements on an Ortho Cytofluorograf, we
would also use saline. There would be a lot of noise after the baseline and
we were adding the stimulous to the sample. Saline reduced this noise so
our calcium signals were more meaningful. We have not noticed any problems
otherwise.

Doug

At 05:09 PM 3/29/2000 EST, you wrote:
>
>Hello Flowers,
>We have found that using distilled/de-ionized water in the sheath is
noisier than
>buffer on our cytometers. After puzzling over the question of contaminants
in the
>water and after some discussion with a few gurus, we have concluded that
the noise
>is not from contaminated water but may result from light bouncing off the
refractive
>index change at the water (sheath): buffer (sample core) interface.
>
>Any thoughts on this?
>
>Alice
>Alice L. Givan
>Englert Cell Analysis Laboratory
>of the Norris Cotton Cancer Center
>Dartmouth Medical School
>Lebanon, New Hampshire NH 03756
>tel 603-650-7661
>fax 603-650-6130
>givan at dartmouth.edu
>
>
>
>
{-----------------------------------------------------------------------}
Douglas Smoot
NIDDK-Navy Transplantation Augmentation Branch
Naval Medical Research Center
AFRRI Building 46, Room 2415
8901 Wisconsin Avenue
Bethesda, MD 20889-5603
voice: 301-295-1843
fax: 301-295-6484



From sasa.sreckovic at bris.ac.uk Sun Apr 2 11:00:24 2000
From: sasa.sreckovic at bris.ac.uk (Dr Sasha Sreckovic)
Date: Sun, 02 Apr 2000 17:00:24 +0100
Subject: PMT for Cy7?
Message-ID: <032ff4059150240INT1@worldonline.co.uk>


Dear Joseph,

I've used APC-Cy7, excited by Spectra-Physics He-Ne 35mW 635nm red laser and
filters: 3-way beam splitter (mirror) -> 710 DMLP -> 780/60 BP.

Anti-mouse CD8 alpha APC-Cy7 gave only 1-log difference, but nice separation
from background (which is lower on red excitation). This was with
red-sensitive Hamamatsu R-3896 PMT and to obtain same mean value on positive
population with R-928 PMT, voltage had to go from 750 to 800! This change
would probably give more difference with something brighter, if possible -
same CD8 was quite bright coupled with PE-Cy5.

Sasha.


************
Dr Sasha Sreckovic
Dept Path & Micro
University of Bristol
University Walk
Bristol, BS8 1TD, UK
Sasa.Sreckovic at bristol.ac.uk
+44-(0)117-928-8606
************



----------
>From: Joseph Webster <J.Webster at centenary.usyd.edu.AU>
>To: Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu>
>Subject: PMT for Cy7?
>Date: Wed, Mar 29, 2000, 3:22 AM
>

>
> We want to try out APC/Cy7 on our FACStar Plus, some clues
> would be helpful from anyone who has used it on a StarPlus
> or Vantage.
>
> We have a red HeNe laser that gives 35-40mW.
> We have ordered Omega filters 710DRLP and 740EFLP to read
> APC and APC/Cy7 in FL4 & FL5.
>
> I believe the standard PMT will probably not be red-sensitive
> enough for Cy7.
> If it won't work, what PMT should I buy?
> Also what's the cheapest source for us in Australia?
>
> What have I missed or forgotten?
>
> Thanks, Joseph.
>
> Joseph Webster, Flow Cytometry Facility,
> Centenary Institute
> Locked Bag No.6, Newtown, NSW 2042, AUSTRALIA.
> Ph: 61-2-9565-6110 Fax: 61-2-9565-6101
> e-mail: J.Webster at centenary.usyd.edu.au
>


From hms at shapirolab.com Sat Apr 1 19:00:57 2000
From: hms at shapirolab.com (Howard Shapiro)
Date: Sat, 01 Apr 2000 19:00:57 -0500
Subject: Quantitation of virus particles
In-Reply-To: <11B42596E849D311B1700000F80824A23FD79A@dasmthefl800.amedd.army.mil>
Message-ID: <4.2.0.58.20000401185253.0097fd50@shell1.shore.net>


Doug Reed wrote:


>Okay, on to the question. We are trying to develop a new assay to quantitate
>virus particles in aerosol samples and tissues using a technique published
>in Biotechniques last year. Essentially, a monoclonal against the virus of
>interest is bound to a bead (in the original paper adenovirus, in our case
>vaccinia). You culture the beads with your sample of interest, wash, then
>follow with a DNA stain (PI or TOTO-1 in the original paper, YOYO-1 in our
>case). By using dilutions of a known concentration (pfu - plaque-forming
>units) of virus, you build a standard curve to compare your unknown with.
>The principle is that you only recognize intact (protein + nucleic acid)
>virus particles since the mAb binds the outer coat but the DNA stain is your
>signal.
>
>So far, so good. You can use the standard curve, but this relates the
>fluorescence simply to pfu.
>
>The question we have is how many intact virus particles are in 1 pfu?
>
>Does anyone have any suggestions for quantitating the actual number of virus
>particles bound to the bead? Is it possible to calculate the number of
>fluorophores that bind to the DNA of the virus and then calculate the amount
>of DNA bound, and hence the number of particles?

Vaccinia are relatively large viruses. The assay you're using already
established that you can stain them with dyes such as TOTO-1 and YOYO-1,
and that there is at least one antibody binding site; one would expect,
viruses being built as they are, that there are several binding sites, and
that one could therefore stain an individual vaccinia virion with at least
a few hundred molecules of YOYO-1 or a similar dye (Pico green might be
even better) and a few dozen molecules of PE-antibody. The
high-sensitivity, slow-flow cytometers first described by me and the Block
group in the late 1970's, and since made and improved on at Los Alamos and
CalTech, should be able to detect both scatter and the fluorescence signals
from individual vaccinia viruses; the CalTech instrument could sort them as
well. As it happens, I now have some funding to look into this, so I
invite interested parties to get in touch.

-Howard





From a.pizzey at ucl.ac.uk Mon Apr 3 03:33:55 2000
From: a.pizzey at ucl.ac.uk (Arnold Pizzey)
Date: Mon, 03 Apr 2000 09:33:55 +0100
Subject: H2DCFDA
In-Reply-To: <200003311113.GAA22274@flowcyt.cyto.purdue.edu>
Message-ID: <3.0.6.32.20000403093355.009376b0@pop-server.bcc.ac.uk>


At 12:05 31/03/00 +0100, you wrote:
>
>Hi!
>
>We are interested in measuring cellular ROS production by the use of
>H2DCFDA in flowcytometry. Company instructions indicate that the dissolved
>reagent has a short stability (few days). The product is provided in 100mg
>size package. Thus, on one hand it is too difficult (and overall too
>harmful) to weight small quantities, and, on the other, if it is necessary
>to dissolve the whole amount for each test, the cost of experiments will be
>too high.
>We would like to know your experience about the storage conditions of
>H2DCFDA, and if anyone has tested the stability of the dissolved reagent.
>Thanks .
>
>Bye
>
>Angela & Anna
>
>Dr. Walter Giaretti
>Laboratory of Biophysics and Cytometry
>National Cancer Institute (IST)
>Largo Rosanna Benzi, n. 10
>16132 Genoa, Italy
>
>Tel. +39-10-5600969
>Fax +39-10-5600711
>E-mail giaretti at hp380.ist.unige.it

Greetings Angela & Anna,

I have used the above reagent (molecular probes D-399) for the measurement
of granulocyte ROS in a whole blood assay. It has been my experience that
the reagent is unstable once dissolved in DMSO; typically we would make it
up on the day of the experiment and discard any unused.

We have been using a 100mg bottle of the above on and off for a few years
now- it never seems to run out and at 130K Lira it couldn't be much
cheaper. Also as far as I can determine, there are no particular safety
issues when handling this substance.

Best regards,

Arnold


_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/
Arnold Richard Pizzey
Department of Haematology
Royal Free and University College London Medical School
98 Chenies Mews
London WC1E 6HX
U.K

voice: +44 0207-209-6234
Fax: +44 0207-209-6222
email: a.pizzey at ucl.ac.uk
_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/


From sasa.sreckovic at bris.ac.uk Sun Apr 2 11:00:05 2000
From: sasa.sreckovic at bris.ac.uk (Dr Sasha Sreckovic)
Date: Sun, 02 Apr 2000 17:00:05 +0100
Subject: Cellquest crashing
Message-ID: <034e22059150240INT1@worldonline.co.uk>


Dear Abby,

Just a tip on freeze-ups. Instead of pulling a plug from the wall its safer
to use 3-key combination: COMMAND/CTRL/POWER-BUTTON for restart, works
always.

Sasha.


************
Dr Sasha Sreckovic
Dept Path & Micro
University of Bristol
University Walk
Bristol, BS8 1TD, UK
Sasa.Sreckovic at bristol.ac.uk
+44-(0)117-928-8606
************



----------
>From: Abby Kelliher <allena at helix.mgh.harvard.edu>
>To: Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu>
>Subject: Cellquest crashing
>Date: Thu, Mar 30, 2000, 10:54 PM
>

>
> Hello -
> I have recently begun having a very annoying problem with my Mac on my
> FacsCalibur. Several times a day, when I try to print I get an error
> message which reads something like: Not enough memory, close some views
> and try again. It is usually the only document open and it is a normal
> (usual file size which for years I have not had this problem with) file
> size. The computer then freezes up. The only way for me to get it back is
> for me to unplug the computer from the wall. I have been working with BD
> for a couple of days on this problem, but we have been unable to solve it
> yet. We have tried: 1)rebuilding the desktop with the extensions off
> 2)rebuilding the desktop with the extensions on 3) throwing out the finder
> preferences and rebuilding the desktop 4) run disk first aid from the Mac
> operating system cd (finds an error everytime I run it and supposedly
> repairs it 5) upped the memory allotment to cellquest 6) upped the disk
> cache
>
> It appears I was having a problem with the network as well, but I don't
> think the two problems are necessarily related. A few minutes ago, the
> worklist manager just quit on its own (while running) and left cellquest
> and the loader manager open but uncontrollable. When I tried a force
> quit, the computer locked up on me. I have had it freeze up on two other
> occasions while acquiring data.
>
> If anyone has any suggestions, I would GREATLY appreciate hearing them. I
> am at my wits end right now.
>
> Thanks in advance!
>
> Abby
>
>
> Abby Kelliher
> Clinical Flow Cytometry Laboratory
> Warren Bldg. Room 112
> Massachusetts General Hospital
> 275 Charles St.
> Boston, MA 02114
>
> 617-726-8487
>
>


From lkrueg at ix.netcom.com Sun Apr 2 08:19:27 2000
From: lkrueg at ix.netcom.com (lori krueger)
Date: Sun, 2 Apr 2000 09:19:27 -0400
Subject: Annexin V help
References: <4.2.0.58.20000327132705.00982bc0@pop.service.ohio-state.edu>
Message-ID: <006001bf9ca6$138746c0$dea4fea9@8skha>


Andrew,
Under some circumstances, I have experienced non-specific staining with
Annexin V-FITC which went away with Annexin V-PE.
Lori
----- Original Message -----
From: Andrew Oberyszyn <oberyszyn.2 at osu.edu>
To: cyto-inbox
Sent: Monday, March 27, 2000 2:48 PM
Subject: Annexin V help


> Hi everybody in Flow land,
> I hope someone can steer me on this one.
> I have a user who is looking at Annexin V- FITC/PI staining of Caco-2
> cells. The problem that I am encountering is that the untreated, Annexin
> V- FITC only stained cells are a log higher than untreated/unstained cells
> (see attached Powerpoint slide). I looked under a flourescent microscope
> and saw considerable surface staining in the Annexin only samples. The
> user is using the Pharmingen kit (5 ul of Annexin V).
> I have not seen this happen (Annexin V so much higher than unstained) in
> other cell lines that I've analyzed. Would it be "legal" to adjust the
> volts to bring the Annexin V Only stained population into the negative
> region or is it more correct to just set the gate as I've indicated on the
> attached slide (marked with an *)?
> Am I missing something obvious? Please help!
>
> Thanks in advance!
> Andy
>
>


----------------------------------------------------------------------------
----


> (:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)
> Andy Oberyszyn, M.S.
> The Ohio State University
> Analytical Cytometry Laboratory
> 416 Comprehensive Cancer Center
> 410 West 12th Avenue
> Columbus, Ohio 43210
> Tel: 614/292-FLOW(3569)
> Fax: 614/292-7335
> E-Mail: cytometry at osu.edu
> Web Page:
> <http://cytometry.med.ohio-state.edu>http://cytometry.med.ohio-state.edu
> (-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-)
>
> "What if the Hokey Pokey is really what it's all about?!?"



From Carol.W.Johnson at ap.pnu.com Mon Apr 3 08:29:35 2000
From: Carol.W.Johnson at ap.pnu.com (Carol.W.Johnson@ap.pnu.com)
Date: Mon, 3 Apr 2000 09:29:35 -0400
Subject: platelet fixation
Message-ID: <852568B6.004A2C9B.00@ap.pnu.com>




I can only provide some experiences with P-selectin expression. The epitope
recognized by many of the anti-P-selectin antibodies are stable to formalins but
labile to alcohols. Paraformaldehyde fixation should work, but may result in a
mixture of permeablized and non-permablized cells. Hence the evaluation could
be a mixture of internal and external P-selectin expression. I assume you are
interested in external expression only? You may be able to gate the external
expression (it will be much lower level than internal expression). I suggest
you may want to look at the cells under a fluorescent microscope before doing
flow on them to get an idea of how many have surface or cytoplasmic staining.

Another problem is that almost any in vitro handling of platelets can activate
them and cause surface expression of P-selectin on a variable percentage of
platelets. You might want to practice and validate the handling of of normal
samples including re-evaluation at different timepoints. Flow cytometry
evaluation of P-selectin expression on platelets can be done but it can be
tricky.

Carol W. Johnson, DVM PhD
Sr. Research Veterinary Pathologist
Pharmacia
Kalamazoo, MI






William Ross <wgr8w at virginia.edu> on 03/31/2000 09:56:57 AM

To: cyto-inbox
cc: (bcc: Carol W Johnson/USKZO/PNU)
Subject: platelet fixation





does anyone no of a good platelet fixation procedure for the study of
selectin expression? It is a study of myocardial infarction patients and
timing the heart attacks is difficult and we're trying to fix the purified
prep for later analysis.

thanks in advance.

Bill Ross

William (Bill) Ross
Director - FACS Core Facility
MR-4, Box G005
University of Virginia School of Medicine
Charlottesville, VA 22908
office (804) 982-1586
fax (804) 924-1221
e-mail:wgr8w at virginia.edu








From JSwiggett at carolinas.org Mon Apr 3 06:40:27 2000
From: JSwiggett at carolinas.org (Swiggett, Jeanene)
Date: Mon, 3 Apr 2000 07:40:27 -0400
Subject: Cellquest crashing
Message-ID: <C229FDA68961D211B99D0008C724FC74EE7E54@dcr-xchg-a.carolinas.org>

Abby, Although the crash/lock-up experiences I have had have been on the
Facstar Plus while running Cellquest. I have found that if the CD player is
running or was run at any time after Cellquest was started I can have a
"freeze". The machine will completely lock-up and I too must shut down from
the wall (in my case - turn off the surge protector) and reboot. This
usually happens when I'm sorting - in other words when you have other BD
functions/programs running in conjuntion with Cellquest. I've also had it
lock up when I've only run Cellquest but I've collected 150 to 200ish files
continuously. Jeanene

Jeanene Swiggett, BS MT(ASCP)SH
General Surgery Research
Cannon Research Center
Carolinas Medical Center
Charlotte, NC
704-355-7269
jswigget at carolinas.org

> -----Original Message-----
> From: Abby Kelliher [SMTP:allena at helix.mgh.harvard.edu]
> Sent: Thursday, March 30, 2000 4:55 PM
> To: Cytometry Mailing List
> Subject: Cellquest crashing
>
>
> Hello -
> I have recently begun having a very annoying problem with my Mac on my
> FacsCalibur. Several times a day, when I try to print I get an error
> message which reads something like: Not enough memory, close some views
> and try again. It is usually the only document open and it is a normal
> (usual file size which for years I have not had this problem with) file
> size. The computer then freezes up. The only way for me to get it back
> is
> for me to unplug the computer from the wall. I have been working with BD
> for a couple of days on this problem, but we have been unable to solve it
> yet. We have tried: 1)rebuilding the desktop with the extensions off
> 2)rebuilding the desktop with the extensions on 3) throwing out the
> finder
> preferences and rebuilding the desktop 4) run disk first aid from the Mac
> operating system cd (finds an error everytime I run it and supposedly
> repairs it 5) upped the memory allotment to cellquest 6) upped the disk
> cache
>
> It appears I was having a problem with the network as well, but I don't
> think the two problems are necessarily related. A few minutes ago, the
> worklist manager just quit on its own (while running) and left cellquest
> and the loader manager open but uncontrollable. When I tried a force
> quit, the computer locked up on me. I have had it freeze up on two other
> occasions while acquiring data.
>
> If anyone has any suggestions, I would GREATLY appreciate hearing them. I
> am at my wits end right now.
>
> Thanks in advance!
>
> Abby
>
>
> Abby Kelliher
> Clinical Flow Cytometry Laboratory
> Warren Bldg. Room 112
> Massachusetts General Hospital
> 275 Charles St.
> Boston, MA 02114
>
> 617-726-8487
>
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From J_Voorn at CLB.nl Mon Apr 3 04:37:50 2000
From: J_Voorn at CLB.nl (Voorn, John)
Date: Mon, 3 Apr 2000 11:37:50 +0200
Subject: platelet fixation
Message-ID: <633112D354E6D111BA540008C724D2640159BA26@clbmx01.clb.nl>


Enclosed a reply by one of our scientists, of course I can not resist the
temptation to inform you that the antibodies which are used are original CLB
clones which are commercially available.
John

Bill,

We have received your mail about platelet fixation.

We are using FACS-analyses also to measure the expression of several
antigens (also P-selectin). To isolate platelets from fixated blood is
described in 1990 by R. Fijnheer et al (Transfusion 30: 20-25, 1990).

One volume of blood was collected into nine volumes of 1% PFA (diluted in
PBS). To make sure that there will be not too much activation due to the
collection a large needle can be used and the first milliliters of blood can
be thrown away. Make sure that during the collection of blood not too much
pressure is forced (flawless collection). Blood was stored at room
temperature.

After one day (we have no experience with longer storage) blood was
centrifuged by a soft spin and the PRP was collected. The isolated platelets
could than be used for FACS-analysis.

I hope that this information will be satisfying, if you have further
questions you can inform us.

Yours sincerely,

Christine Kramer
Technician of Blood Transfusion Technology
CLB, Amsterdam

On behalf:
Dr. D. de Korte
Head of laboratory of Blood Transfusion Technology

John Voorn CLB Reagents



Productmanager Plesmanlaan 125
j_voorn at clb.nl <mailto:j_voorn at clb.nl> 1066CX
AMSTERDAM
the Netherlands
tel +31-(0)20 5123246
fax +31-(0)20 5123570 web site www.clb.nl <www.clb.nl>






-----Original Message-----
From: William Ross [SMTP:wgr8w at virginia.edu]
Sent: vrijdag 31 maart 2000 16:57
To: Cytometry Mailing List
Subject: platelet fixation


does anyone no of a good platelet fixation procedure for the study
of
selectin expression? It is a study of myocardial infarction
patients and
timing the heart attacks is difficult and we're trying to fix the
purified
prep for later analysis.

thanks in advance.

Bill Ross

William (Bill) Ross
Director - FACS Core Facility
MR-4, Box G005
University of Virginia School of Medicine
Charlottesville, VA 22908
office (804) 982-1586
fax (804) 924-1221
e-mail:wgr8w at virginia.edu



From shenww at public3.sta.net.cn Mon Apr 3 04:05:16 2000
From: shenww at public3.sta.net.cn (Weiwen Shen)
Date: Mon, 3 Apr 2000 17:05:16 +0800
Subject: About platelet activation
Message-ID: <001a01bf9d4b$df635400$1f0d813d@oemcomputer>

Dear Sirs,
I am novice in flow cytometry, though know it for a long time. But as you know, in China, no good teacher is available for me in this field. I learned much here and thank all the scientists here to supply such knowledge. Actually I am sales rep. of flow cytometry reagent in China, and we distribut PharMingen, BD, etc. You know, scientists in China as in other developing countries, they need more help than in developed countries. As sales rep. I want to do my best to make them pleased. So I will ask many questions from them, if anyone can help, we are appreciated. Thank you again for your knowledgeable information.
Question today is:
1. How to detect to activation status of platelets? The customer need to know whether his patients treated with a kind of Chinese traditional medicine have a reduced level of platelet activation. The antibodies available here are anti-human CD41a, CD41b, CD61, CD62P and PAC-1, but someone said that some clones of anti-human CD41a antibodies can not discriminate the activated and unactivated form of GPIIb/IIIa. So I thought that we need such an antibody together with FSC to gate out the platelets and analyze the platelets with other parameter. So if you are experienced in this kind of detection, please give me your suggestion. Aslo, the customer need to set a rat model, so he need anti-rat reagents too, how to choose the antibodies?
2. Does anyone know a good review or reference on the relationship of cell surface markers and tumor?
3. Two parallel experiments detceted CD3/CD16+56 and CD3/CD4/CD8, the CD3 positive percent is different in the same gated cell population, what may cause such a difference?
Thank all the scientists here.
Sorry for my poor English.

Weiwen Shen
M.S.
Jingmei Biotech Shanghai

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From amr_farouk_eg at yahoo.com Mon Apr 3 12:27:41 2000
From: amr_farouk_eg at yahoo.com (Amr Farouk)
Date: Mon, 3 Apr 2000 10:27:41 -0700 (PDT)
Subject: Annexin V?????????
Message-ID: <20000403172741.326.qmail@web4205.mail.yahoo.com>


Hello every body

I wounder if there any one have tried to quantify
apoptosis in solid tuomrs using annexin V-FITC by flow
cytometry?

Amr Farouk
Ph.D student
Faculty of medicine
Mansoura University
Egypt




__________________________________________________
Do You Yahoo!?
Talk to your friends online with Yahoo! Messenger.
http://im.yahoo.com


From cali at liai.org Mon Apr 3 18:56:29 2000
From: cali at liai.org (Chris lena)
Date: Mon, 3 Apr 2000 15:56:29 -0800
Subject: Antibody again
Message-ID: <v04003a00b50ee007b425@[172.20.0.115]>


I guess I should have indicated that this is to be anti-mouse (LY3.1)
>Hi List,
>Does anyone know where to find a conjugated anti-CD8bchain antibody which
>is specific to LY3.1? It would be a huge help. Thank you.
>chris
>
>
>



Chris Lena
Laboratory Technician
La Jolla Institute For Allergy and Immunology
phone:(858)678-4536
fax:(858)678-4595
cali at liai.org
http://www.liai.org

_ _
(_)-(_)
\"/
=V=






From Brad_Billman at bd.com Tue Apr 4 10:34:55 2000
From: Brad_Billman at bd.com (Brad_Billman@bd.com)
Date: Tue, 4 Apr 2000 10:34:55 -0500
Subject: Position Offered at Becton Dickinson Biosciences
Message-ID: <200004041535.KAA20772@flowcyt.cyto.purdue.edu>


Becton Dickinson Biosciences is seeking an individual for sales of technical
products to the research industry. The potential candidate should have a
scientific degree and training, experience in Flow Cytometry and/or Cell
Culture as well as a desire to support the scientific community in furthering
their research through innovative products and offering technical support.
Some travel is required. Position will be based out of Southern Ohio or
Western Kentucky.

Please FAX cv/resume to (972)424-4268.





From pplett at iupui.edu Mon Apr 3 13:20:08 2000
From: pplett at iupui.edu (Plett, P Artur)
Date: Mon, 3 Apr 2000 13:20:08 -0500
Subject: Mouse lymphocyte separation
Message-ID: <87BAA98D14EED311953400508B8BED440FD990@iupuimbx08.uits.iupui.edu>


We used Ficoll 1.083, from Sigma, in my old lab (I don't know the cat.
number) which works great with mouse lymphocytes, spleen or blood.
Artur

> ----------
> From: Cathryn Broderick
> Sent: Thursday, March 30, 2000 13:15
> To: Cytometry Mailing List
> Subject: Mouse lymphocyte separation
>
>
> Does anyone know of a method for separating mouse
> lymphocytes from cell suspension, similar to human
> lymphocytes and histopaque?? That is, is there a
> commercial product available specifically for mouse
> lympocytes, or which protocols are people currently using
> instead?
>
> Thanks for any help...
>
> Cathryn
>
> ----------------------
> Cathryn Broderick
> c.broderick at abdn.ac.uk
>
>
>
>


From s9070419 at pop3.unsw.edu.au Mon Apr 3 20:15:37 2000
From: s9070419 at pop3.unsw.edu.au (Robert Nordon)
Date: Tue, 4 Apr 2000 11:15:37 +1000
Subject: HLA typing by flow
Message-ID: <020a01bf9dd3$a7308b60$0b59ab95@gsbme.unsw.edu.au>

Dear flowers

A reference for Human HLA typing by flow?

Many thanks


Robert Nordon
Research Fellow
Graduate School of Biomedical Engineering
University of New South Wales, 2052.
Tel: 61-2-9385-3906
Fax: 61-2-9663-2108
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From bigos at stanford.edu Mon Apr 3 16:25:14 2000
From: bigos at stanford.edu (Marty Bigos)
Date: Mon, 03 Apr 2000 14:25:14 -0700
Subject: PMT for Cy7?
In-Reply-To: <01JNOGYLUCIQ9AN68T@wehi.edu.au>
References: <01JNOGYLUCIQ9AN68T@wehi.edu.au>
Message-ID: <v04210114b50ebb64cdd8@[171.65.21.136]>


Frank-

The 636-10 is GaAs type PMT. Although they have better spectral
responses in the red than the 3896, 928, 1477 types, their gain is an
order of magnitude less. Thus, the match may not be optimal for the
preamp system gains that BD uses. Have you (or anyone else reading
this) had any experience with these PMTs?

Marty

>Joseph,
>
>As of May 1996, Hamamatsu's recommendation was R636-10. Your Sydney
>supplier is Stantron Australia P/L, contact Stan Hill.
>ph: (02) 9894 2377, fax: (02) 9894 2386. Price in 1996 was $AUS2550.
>
>I haven't tried APCCy7 on our FACStar+ but it goes on the MoFlo with a
>35nW HeNe so there's no reason it should be difficult. I also don't
>know how much the extreme red PMT helps but if it will save $2.5k why
>not try it first with the existing PMTs.
>
>Frank.
>
>Frank Battye, \ / < The Cytometry Laboratory
>Dora Kaminaris, \__/ <<<<< The Walter & Eliza Hall Institute
>Viki Lapatis, ------!!<<<<<<<< Victoria 3050, AUSTRALIA
>Jennie Parker and /!!\ <<<<< ph: 61_3_9345 2540 fax: 61_3_9347 0852
>Catherine Tarlinton o !! \ < in:: "facs_copy at wehi.edu.au"



From justin-fishbaugh at uiowa.edu Mon Apr 3 13:49:55 2000
From: justin-fishbaugh at uiowa.edu (Justin Fishbaugh)
Date: Mon, 03 Apr 2000 13:49:55 -0500
Subject: CPU control card for EPICS 753
Message-ID: <3.0.32.20000403134953.00686194@flocyt.int-med.uiowa.edu>


Greetings,

We have worn out the thumb wheel switches on the CPU control card that
control the X and Y axis for live oscilloscope dot plots of our Coulter
EPICS 753. A wild goose chase to find two switches from the switch
manufacturer has so far turned up nothing. If some sympathetic soul has a
CPU control card for an EPICS 750 series MDADS II laying around, we would
be grateful for its use. Thanks.


------------------------------------------------
| Justin Fishbaugh |
| University of Iowa |
| Flow Cytometry Facility |
| 48 EMRB |
| Iowa City, IA 52242 |
| |
| justin-fishbaugh at uiowa.edu |
| http://www.medicine.uiowa.edu/flowcytometry/ |
------------------------------------------------


From FACS_COPY at wehi.edu.au Tue Apr 4 00:41:24 2000
From: FACS_COPY at wehi.edu.au (FACS_COPY@wehi.edu.au)
Date: Tue, 04 Apr 2000 15:41:24 +1000
Subject: PMT for Cy7?
Message-ID: <01JNUB3QY7DY9ANAVT@wehi.edu.au>


Marty,

> The 636-10 is GaAs type PMT. Although they have better spectral
> responses in the red than the 3896, 928, 1477 types, their gain is an
> order of magnitude less. Thus, the match may not be optimal for the
> preamp system gains that BD uses. Have you (or anyone else reading
> this) had any experience with these PMTs?

In 1996, we specified a type R636-10 for our MoFlo before the specs on
the R3896 were available to us. It was supplied as Cytomation type
H957-11. Hamamatsu's specs (http://www.hpk.co.jp/HP2E/main.html) show
cathode radiant sensitivities at 800nm of about 15, 40 and 60 mA/W for
types R928, R3896 and R636-10 respectively. I'm persuaded that the
less than 30% cathode sensitivity improvement of the R636-10 over the
R3896 is not worth the lower gain (about 20-fold). On the other hand,
200V more PMT volts should give a decade more gain. In fact we
typically run our R636-10 PMT at 750V which is 100-200V more than the
FITC and PE detectors.

You can see an actual result at
http://www.wehi.edu.au/cytometry/FCMexamples.html#apccy7 but we
haven't done the comparison with any other PMT.

Frank.

Frank Battye, \ / < The Cytometry Laboratory
Dora Kaminaris, \__/ <<<<< The Walter & Eliza Hall Institute
Viki Lapatis, ------!!<<<<<<<< Victoria 3050, AUSTRALIA
Jennie Parker and /!!\ <<<<< ph: 61_3_9345 2540 fax: 61_3_9347 0852
Catherine Tarlinton o !! \ < in:: "facs_copy at wehi.edu.au"




From Eak at medicine.wisc.edu Mon Apr 3 11:27:21 2000
From: Eak at medicine.wisc.edu (Becky Kelly )
Date: Mon, 03 Apr 2000 11:27:21 -0500
Subject: Postdoctoral postion
Message-ID: <s8e88030.041@gw.medicine.wisc.edu>


A postdoctoral position is available in the University of Wisconsin Department of
Medicine, Pulmonary and Critical Care Section, to study the immunological mechanisms of
airway remodeling in asthma. The candidate will use molecular and cellular immunology
methods to study the interactions among airway T cells, eosinophils, and fibroblasts,
and signaling pathways and cell cycle control of airway fibroblasts. Experience in
immunology, cell biology, signal transduction, and molecular biology is desirable.
Send statement of interest, curriculum vitae, and addresses of three references to:
Nizar N. Jarjour, M.D., University of Wisconsin School of Medicine, 600 Highland Ave.,
Madison, WI., 53792.






Elizabeth A. (Becky) Kelly, Ph.D.
University of Wisconsin, Department of Medicine
600 Highland Avenue, RM H6/380
Madison, WI 53592
(608) 263-3253 (office)
(608) 263-9144 (lab)
(608) 263-3746 (FAX)




From sasa.sreckovic at bristol.ac.uk Tue Apr 4 10:03:47 2000
From: sasa.sreckovic at bristol.ac.uk (Dr Sasha Sreckovic)
Date: Tue, 04 Apr 2000 16:03:47 +0100
Subject: CellQuest crashing
Message-ID: <200004041504.QAA04592@eis.bris.ac.uk>

Hi All,

I really regret that I've hit the "Reply to All" button (sometimes I do use
only "Reply", believe it or not!) when I said this:

> Dear Abby,
>
> Just a tip on freeze-ups. Instead of pulling a plug from the wall its safer
> to use 3-key combination: COMMAND/CTRL/POWER-BUTTON for restart, works
> always.
>
> Sasha.

as I've received dozens of messages proving me wrong, few very examples:

> ........Well, nearly always! Sometimes you just have to pull the plug.

> Wrong! Does not work always! (As described in the Macintosh guide)
> Sometimes even pressing the power button on the front of the computer does
> not help! Then there is nothing else to do than pulling the plug!

Some guys also presumed that my Macs are especially prone to crashing, but
CellQuest didn't freeze in here since Quadras. Formatting my Macs from time
to time with "ZERO ALL DATA" (after backup of course) does them really good,
even more than Desktop rebuilding and disk driver updating.

Now, I had to go and check with a few Mac Gurus (thanks to Stefan Mueller
and Apple Support) and overall opinion is that older PowerPCs and earlier
Macs do not always restart on this (Apple Support said only with MacOS 8 or
later). Anyway, sometimes it's better to leave it disconnected (pull a plug)
for one minute - for memory to empty, just in case of resident conflicts
reappearing.

It could be that people assign to much memory to CellQuest and there is not
enough memory left for printing. Printer Monitor is going to take as much as
it can get from available memory but if there is less then needed for
spooling this problem may occur.

This was valuable lesson, I'll be more careful on Mac issues, it seems there
are a lot of guys out there playing with it more than me!

Thank you all,
Sasha.


----------------------
Dr Sasha Sreckovic
Dept Path & Micro
University of Bristol
University Walk
Bristol, BS8 1TD, UK
Sasa.Sreckovic at bristol.ac.uk
Tel +44-(0)117-928-8606
Fax +44-(0)117-928-7896





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From allena at helix.mgh.harvard.edu Mon Apr 3 15:51:37 2000
From: allena at helix.mgh.harvard.edu (Abby Kelliher)
Date: Mon, 3 Apr 2000 16:51:37 -0400
Subject: FACSComp QC
Message-ID: <01BF9D8C.E1635930.allena@helix.mgh.harvard.edu>


We are a clinical lab and we started running FACSComp once per day, but
found it to be unnecessary. We were able to document that our voltages did
not change much at all from week to week, so we now run FACSComp once per
week, unless 1) the instrument is cleaned, 2) it is serviced, 3) there is a
noticeable problem. It is good for calibrating the instrument, but most
problems I see are not diagnosed by FACSComp, and I don't recommend relying
on it for that purpose.


Abby Kelliher
Clinical Flow Cytometry Laboratory
Warren Bldg. Room 112
Massachusetts General Hospital
275 Charles St.
Boston, MA 02114

617-726-8487


-----Original Message-----
From: CJett [SMTP:CJett at Compucyte.com]
Sent: Friday, March 31, 2000 12:49 PM
To: Cytometry Mailing List
Subject: RE: FACSComp QC


CJ> We were originally running FACSComp at the beginning of each day...but
later found our stability was good enough to go to a weekly check
up.....but
you have to be vigilant for voltage creep on your laser..once you get
around
3000 hrs the 488nm argon can start to drop off and get noisy. Of course a
clinical lab will not want to do this..once a day, keeps the FDA away



>>> Tami Rosario <trosario at cellmate.cb.uga.edu> 03/29/00 12:55PM >>>

I have a question for those of you who run FACSComp on the FACSCaliber
for QC. Do you run this each time you start up the cytometer or only
occasionally. I have been told that I should run FACSComp each day that
I run samples, but the beads are fairly expensive. Any alternatives?

Thanks,
Tami





From ewieder at gladstone.ucsf.edu Tue Apr 4 12:57:41 2000
From: ewieder at gladstone.ucsf.edu (Eric Wieder)
Date: Tue, 4 Apr 2000 10:57:41 -0700
Subject: Job Postings
Message-ID: <v04011702b50fdd1bc4b5@[128.218.162.133]>

The Core Flow Cytometry Lab at the Gladstone Institute of Virology and
Immunology/UCSF is looking for a Director (see job description below). Two
positions are also open at the Research Associate level for individuals
with flow experience. If interested in learning more about the positions
and the Lab, please contact and/or send CV to:

1) Mike McCune, 415-695-3828 (phone), 415-826-8449 (fax),
mmccune at gladstone.ucsf.edu
2) Eric Wieder, 415-695-3832 (phone), 415-826-8449 (fax),
ewieder at gladstone.ucsf.edu


FACS CORE DIRECTOR - JOB DESCRIPTION

1. Instrumentation expert - The Core Director will be responsible for the
maintenance and optimization of two cell sorters (a FACSVantage and a
Cytomation MoFlo) and two bench-top cell analyzers. In addition, the Core
Director will be responsible for maintaining three microscopes - a
fluorescence microscope (Leitz), a laser capture microdissection scope, and
a time-lapse fluorescence video microscope.

2. FACS techniques expert - The Core Director will provide
expertise/protocols of the latest in flow cytometric techniques to the
members the Gladstone Institutes and other users of the Core. The Director
will assist colleagues in the appropriate interpretation and analysis of
FACS data. S/he will also keep up with the literature and attend
scientific conferences and relay relevant new methods/findings to other
members of the institutes.

3. FACS trainer - The Core Director will instruct users in the basics of
running a flow cytometer and of analyzing flow cytometric data.

4. Management - The Core Director will interact with other Gladstone
Institutes departments (i.e., facilities management and computer support)
to ensure the smooth operation of the core and of the data flow off of the
instruments. Furthermore, the Core Director will hire and train assistants
as needed to insure that needs for FACS support are being met. The Core
Director will devise and institute policies to ensure that access to the
machine for all users is maintained and conflicts between users resolved.
Finally, the Core Director will manage access to the machines to insure
that appropriate access is provided and that safety is not compromised.

5. Liaison to the FACS community at large - The Core Director will interact
with flow cytometer manufacturers, reagent manufacturers, and other flow
cytometry cores to ensure that members of the Gladstone Institutes are
up-to-date on the most current technologies.
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From beckk at ukl.uni-freiburg.de Wed Apr 5 08:56:43 2000
From: beckk at ukl.uni-freiburg.de (Kerstin)
Date: Wed, 5 Apr 2000 14:56:43 +0100
Subject: Ntera2
Message-ID: <v04003a01b510f4f9ce3a@[193.196.203.101]>


hello everybody,
This is not a really flow-cytometry question, but I hope someone can answer
this question too.
Does anyone has experiences with the cell-line Ntera 2 clone d1? My problem
is that this cells grow very slowly and when I splitt the cells they will
die. I used different concentrations of trypsin or cellscrapers but
everytime the cells died after these procedures. What can I do ?
Thanks
Kerstin

*****************************************
Kerstin Beck
Institut f?r Medizinische Mikrobiologie und Hygiene
Abteilung Virologie
Hermann-Herder-Strasse 11
D-79104 Freiburg
Telefon : +49-(0)761-203-6646
Fax : +49-(0)761-203-6603
beckk at ukl.uni-freiburg.de
*****************************************




From LBarsky at chla.usc.edu Tue Apr 4 14:45:49 2000
From: LBarsky at chla.usc.edu (Barsky, Lora)
Date: Tue, 4 Apr 2000 12:45:49 -0700
Subject: Attn: BD LSR users
Message-ID: <C11866CCBDDBD1119EAD00A0C9AF9AB906997E25@nt_ms31>


Hello,

I found a couple of replies to the purdue site regarding the new BD LSR, but
I'm hoping to hear from more individuals who have already gotten their BD
LSRs up and running. Is the instrument easy to use, if you're not an
"expert"? Could students easily operate the instrument? Is alignment easy
to do with the calibration beads? And the optics, is it a matter of
switching only bandpass filters or is there dicroic involvement? Does
anyone have a 3-color LSR? And finally, what type of lab do you have it in,
a Core, private lab, etc.?

Thanks for the time,
Lora Barsky


From virginia.litwin at bms.com Tue Apr 4 16:02:20 2000
From: virginia.litwin at bms.com (Virginia M Litwin)
Date: Tue, 04 Apr 2000 17:02:20 -0400
Subject: elastase in neurtophils
Message-ID: <38EA585C.17F563F3@bms.com>

Does anyone know of a method to measure elastase in neutorphils?
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From virginia.litwin at bms.com Tue Apr 4 15:25:48 2000
From: virginia.litwin at bms.com (Virginia M Litwin)
Date: Tue, 04 Apr 2000 16:25:48 -0400
Subject: anti-B7.1 B7.2 mAbs
Message-ID: <38EA4FCC.A32621A8@bms.com>

Is anyone aware of some anti-B7.1 or anti-B7.2 mAbs which DO NOT
inhibit CTLA4 binding?
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From Doug.Reed at DET.AMEDD.ARMY.MIL Tue Apr 4 11:59:45 2000
From: Doug.Reed at DET.AMEDD.ARMY.MIL (Reed, Doug S Dr USAMRIID)
Date: Tue, 4 Apr 2000 12:59:45 -0400
Subject: Quantification of virus particles - followup
Message-ID: <11B42596E849D311B1700000F80824A23FD7AD@dasmthefl800.amedd.army.mil>


Thanks to everyone who has responded so far (and to those who might still
respond). I think the best solutions I've seen so far were Howard Shapiro's
solution of using anti-vaccinia antibodies (PE conjugated) and (even better,
although technically more difficult) putting various pfu concentrations of
virus under an electron microscope and counting.

Some of you commented that you weren't sure that the pfu/particle ratio
wasn't that important. At this point it is unknown what effect it has - but
we have routinely seen viable bacteria counts by flow that are 1-2 logs
higher than the colony counts after aerosol sprays. At this point it is
interesting information, but the relevance is unclear.

Howard - you are right, we could count vaccinia directly by flow given the
large size of poxviruses. I am attempting to develop the beads as a more
generally adaptable technique to the wide variety of viruses we study here -
not just vaccinia but also the more exotic filoviruses which are
considerably smaller. Currently most researchers use vero plaque assays to
determine the pfu of the virus, which is certainly a proven technique but
requires considerable time and effort whereas the beads could be done the
same afternoon as the spray.

Many thanks again to all those who replied!

Douglas S. Reed, Ph.D.
Microbiologist
Department of Aerobiology and Product Evaluation
Division of Toxinology and Aerobiology
U.S. Army Medical Research Institute of Infectious Disease
1425 Porter St. Ft. Detrick
Frederick, MD 21702-5011
301-619-6728
301-619-2541 fax
Doug.Reed at det.amedd.army.mil



From geoff at sparc.brc.ubc.ca Tue Apr 4 13:44:18 2000
From: geoff at sparc.brc.ubc.ca (Geoffrey Osborne)
Date: Tue, 4 Apr 2000 11:44:18 -0700
Subject: LARGE CELL SORT
In-Reply-To: <s8e4bdcd.006@gwsmtp.ohsu.edu>
Message-ID: <v03110702b50fe03e6c1d@[137.82.2.113]>


Hi Randall,
My experience with large nozzle sorting has been with the mostly
the 300um and occasionally the 400um nozzle. My advice is to obtain as
"clean" a preparation of the particles of interest as possible. While this
may seem obvious, the importance of maintaining cells in a single cell
suspension and not having them stick together is critical in obtaining a
stable stream breakoff. This is often difficult due to the effect of
gravity on large particles in suspension, as clumps of 2 or more cells
really become a problem.
On the Vantage SE I'm unable to sort really large stuff (~200um
organisms from termite guts), as it seems to be beyond the normal rules of
thumb that one would normally use with respect to particle size and nozzle
selection, but particles up to about 100um are "do able". For your
particle size you may actually get better stability from the system by
using the 400 over the 300um nozzle. For the 400um nozzle I'd be looking at
about 1000hz ddf, 2PSI and slow flow rate. Block off any other waste
aspiration that you've got on the system as you want to draw as much waste
from the main aspirator as possible. Remember also that you may need to use
the different obscurator bars, and change ND filters on the FSC diode too.
My last tip is be patient, and persist. Hope some of this helps
Geoff


>Dear All,
>
>We have some large cells (70-80um) to sort on our new FACSVantage, and
>have no experience
>with cells of this size. Before we run through gallons of sheath, we would
>like to
>hear of your experience with large cell/large tip sorting. It looks like
>the 300um
>tip is recommended for this size cell.
>
>The Vantage is running well on normal 70um tip for blood and cultured
>cells. (Given the
>problems that occur with a new machine).
>
>All advice Welcome!
>
>Thanks
>
>Randall Smith
>Oregon Health Sciences University
>Flow Cytometry Core Facility
>Portland, Oregon


=====================================================
Geoffrey Osborne
Manager, Multi User Flow Cytometry Facility
Biomedical Research Centre,
2222 Health Sciences Mall,
University of British Columbia
Vancouver B.C V6T 1Z3 CANADA
Email: geoff at brc.ubc.ca
Phone: 604 822 7838
Surfing the Web?: Try http://jcsmr.anu.edu.au/facshome.html or
http://www.cytometry.brc.ubc.ca
=====================================================




From neumann at santacasa.tche.br Wed Apr 5 08:08:57 2000
From: neumann at santacasa.tche.br (Jorge Neumann)
Date: Wed, 5 Apr 2000 10:08:57 -0300
Subject: HLA typing by flow
Message-ID: <01bf9f00$19e026c0$0100090a@ipd1.iscmpa>

Dear Robert,

As far as I know, only B27 is available for routine typing. I have been using it for years and it is a good reagent for screening. The "positives" need confirmation by other methods due to several cross reactions. I use the monoclonal from One Lambda that is very similar to the one from BD.
Regards,

Jorge Neumann
Santa Casa Hospital
Porto Alegre
Brasil

-----Mensagem original-----
De: Robert Nordon <s9070419 at pop3.unsw.edu.au>
Para: Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu>
Data: Ter?a-feira, 4 de Abril de 2000 19:35
Assunto: HLA typing by flow


Dear flowers

A reference for Human HLA typing by flow?

Many thanks


Robert Nordon
Research Fellow
Graduate School of Biomedical Engineering
University of New South Wales, 2052.
Tel: 61-2-9385-3906
Fax: 61-2-9663-2108
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From jhendrik at server.nybc.org Wed Apr 5 17:27:29 2000
From: jhendrik at server.nybc.org (Jan Hendrikx)
Date: Wed, 05 Apr 2000 18:27:29 -0400
Subject: Applescript and Flowjo?
Message-ID: <p04310102b5116dd7c984@[192.94.249.197]>


Dear Flowers,

Has anybody succeeded in scripting Flowjo using Applescript? It would
be great if that were possible!!

Kind regards,

Jan
--




****************************************************************
P. Jan Hendrikx
New York Blood Center
Department of Stem Cell Biology
310 East 67th Street
New York 10021 NY

Jan_Hendrikx at Server.Nybc.Org
Tel.: +1-212-570-3345
Fax: +1-212-570-3352
****************************************************************


From tylee at itis.com Wed Apr 5 18:49:58 2000
From: tylee at itis.com (tylee)
Date: Wed, 5 Apr 2000 18:49:58 -0500
Subject: elastase in neurtophils
Message-ID: <01bf9f59$a6b74e80$2e96a5d8@triss2>


Virginia,

I have not tried this; but, you micht consider looking at this possibility.
http://www.phiphilux.com/elas1.htm
I think this is suppose to be a cell permeable substrate that changes
fluorescent properties upon protease cleavage.
Ty Lee


-----Original Message-----
From: Virginia M Litwin <virginia.litwin at bms.com>
To: cyto-inbox
Date: Wednesday, April 05, 2000 5:12 PM
Subject: elastase in neurtophils


>Does anyone know of a method to measure elastase in neutorphils?
>



From dcoder at u.washington.edu Wed Apr 5 16:08:10 2000
From: dcoder at u.washington.edu (David Coder)
Date: Wed, 5 Apr 2000 14:08:10 -0700
Subject: Attn: BD LSR users
References: <C11866CCBDDBD1119EAD00A0C9AF9AB906997E25@nt_ms31>
Message-ID: <004401bf9f43$0c246440$a3725f80@immunol.washington.edu>


I've had the second beta instrument that was shipped in October of last year.
It's been in a multi-user facility since then.

Performance: boringly stable
Alignment: there is none (good since it's not simple)
Optics: we only swap bandpass filters in front of the PMTs
Ease of use: if someone can use CellQuest, they can run the instrument

But, for complex things (calcium flux with indo-1, DNA (DAPI or Hoechst) with
three or four surface markers, it's important that an experience person setup
the application and make sure that the configuration is correct and the detector
setup and amplifications are appropriate. After that, small changes can usually
be done by the user. Quite naive users are able to run dual laser,
multiparameter experiments and get good results.

Most applications can be done as comes out of the box. If you'll be changing
dichroics, new holders would be needed (I've made some modifications). If the
initial alignment is good, then positioning the dichroic has limited degrees of
freedom as far as realignment is concerned.

Dave

**********************************
David M. Coder, Ph.D.
Director, Cell Analysis Facility
Dept. of Immunology
Univ. of Washington School Medicine
Box 357650
Seattle WA 98195-7650

tel. 206-685-3014
fax. 206-543-3480
email: dcoder at u.washington.edu

----- Original Message -----
From: Barsky, Lora <LBarsky at chla.usc.edu>
To: cyto-inbox
Sent: Tuesday, April 04, 2000 12:45 PM
Subject: Attn: BD LSR users



Hello,

I found a couple of replies to the purdue site regarding the new BD LSR, but
I'm hoping to hear from more individuals who have already gotten their BD
LSRs up and running. Is the instrument easy to use, if you're not an
"expert"? Could students easily operate the instrument? Is alignment easy
to do with the calibration beads? And the optics, is it a matter of
switching only bandpass filters or is there dicroic involvement? Does
anyone have a 3-color LSR? And finally, what type of lab do you have it in,
a Core, private lab, etc.?

Thanks for the time,
Lora Barsky



From kharkins at iastate.edu Wed Apr 5 17:40:30 2000
From: kharkins at iastate.edu (Kristi Harkins)
Date: Wed, 05 Apr 2000 17:40:30 -0500
Subject: sample/sheath ratio after sort
Message-ID: <200004052240.RAA26014@mailhub.iastate.edu>


Hi cytometry fans,

I have a client that wants to know how much of the drop volume will be
taken up by the sample fluid (I need a general estimate). We will be using
an ELITE sorter (12 PSI) with 100 micron sort sense tip to sort. To make my
life easier, has anyone calculated the sample/sheath ratio after sorting at
a specific sample input pressure? I thought about running a concentrated
blue dye and sorting under conditions similar to the eventual client sort
and then measuring the OD in a spec original sample vs. sorted sample. Has
anyone tried this and was it possible to see any absorbance in the diluted
sort sample? Looking forward to your replies,

Kristi Harkins



From Sciex at aol.com Wed Apr 5 17:12:59 2000
From: Sciex at aol.com (Sciex@aol.com)
Date: Wed, 5 Apr 2000 18:12:59 EDT
Subject: Peptide phage library screening by Flow
Message-ID: <90.2a2aaa4.261d146b@aol.com>



In a message dated 4/3/00 4:53:31 PM, rr5845 at hotmail.com writes:

<<
Hi
We use the elisa technique succesfully in our laboratories to detect phage
bound to the leukemic cells. We use the monoclonal mouse anti-M13 antibody
conjugated with HRP to detect the bound phage. But our repeated attepts to
detect bound phage by flow has failed. I have used Mouse anti-M13
unconjugated primary antibody followed by using a anti mouse goat secondary
which is conjugated to FITC. A couple of papers have shown bound phage
detection by using a sheep anti-M13 antibody followed by secoundary
conjugated with FITC with success, But the company Parmacia stopped
production of this antibody. The company has not tried out the mouse anti
M13 primary to check phage bound to cell by flow. So i would be very
grateful if anybody could give me information on protocols tried out or to
use or information of antibodies that may be used for detection of the bound
phage by flow. I have also tried out anti HRP antibody against the anti-M13
HRP primary that we use for the ELISA but still could not show the detection
of phage bound to the cells.
Thanks
Raj
____ >>

Raj,
Exalpha Biologicals, Inc. (800. 395. 1137 or 617. 445. 6463) has an anti M13
antibody that is biotin conjugated. We haven't tested it in flow yet but it
may be worth a try - it's not the Pharmacia clone so who knows.
Regards,
John


From craig.turner at nbs.nhs.uk Thu Apr 6 07:37:21 2000
From: craig.turner at nbs.nhs.uk (craig.turner@nbs.nhs.uk)
Date: Thu, 6 Apr 2000 13:37:21 +0100
Subject: complement binding assay
Message-ID: <TFSLGRMO@nbs.nhs.uk>


Does anyone have experience of assaying platelet associated complement ? I am
looking for an antibody to use in order to assess the level of complement binding to
platelets. Should I target C3 or the C5-9 neo-epitope?

Craig Turner


From nadeem at bart.rprc.washington.edu Wed Apr 5 12:25:51 2000
From: nadeem at bart.rprc.washington.edu (Nadeem Sheikh)
Date: Wed, 5 Apr 2000 10:25:51 -0700
Subject: Complement lysing anti macaque CD4/CD8
Message-ID: <004e01bf9f23$fd49b0c0$41e08e8c@rprc.washington.edu>

Does anybody know of (or have) anti CD4 or 8 capable of complement lysis of macaque CD4 CD8 cells ?
We currently use clone G10-1 for CD8 complement lysis. Any info/help would be greatly appreciated !!


Nadeem Sheikh,
****************************************************************************************
RPRC Department of Pharmaceutics
University of Washington University of Washington
Box 357331 Box 357610
Seattle, WA 98195 Seattle, WA 98195

Correspondence
3000 Western Avenue
Seattle, WA 98121
Voice : +1 206 221 2356
Fax : +1 206 956 0573
e-mail : nadeem at bart.rprc.washington.edu
http://www.rprc.washington.edu/
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From Eileen.Gardenhire at aventis.com Thu Apr 6 17:39:11 2000
From: Eileen.Gardenhire at aventis.com (Eileen.Gardenhire@aventis.com)
Date: Thu, 6 Apr 2000 17:39:11 -0500
Subject: Is there a difference between CD34 and CD34+?
Message-ID: <AFF2E1D861D8D111A34100A0C9AF9909015760F8@brwsmxsusr07.brw.hmrag.com>


I am a data manager for a pharmaceutical company and have the simple
question what is the meaning of the + in CD34+? and is CD34 lymphocytes the
same as CD34+ lymphocytes?

Many thanks,

> Eileen Gardenhire
> Project Data Manager
> Aventis
> PO Box 6800
> Rt. 202-206
> Bridgewater, NJ 08807-0800
> email: eileen.gardenhire at aventis.com
> tele.: 908-231-2464
> fax: 908-231-2136
>
>


From rose_hughes at hgsi.com Thu Apr 6 15:04:26 2000
From: rose_hughes at hgsi.com (rose_hughes@hgsi.com)
Date: Thu, 6 Apr 2000 16:04:26 -0400
Subject: anti-hamster ab's
Message-ID: <OFD9F48B9F.A1D86350-ON852568B9.006DE7BF@hgsi.com>


Dear Flowers,

I am inquiring about a source of anti-hamster antibodies. Any leads will
be appreciated.

Rose Hughes, B.S. CT(ASCP),(IAC),CLsp(CG)
Flow Cytometry Lab
Human Genome Sciences, Inc.
9410 Key West Ave.
Rockville, MD 20850
(301) 309-8504 ext. 2074




From David.McFarland at mcmail.vanderbilt.edu Thu Apr 6 15:23:54 2000
From: David.McFarland at mcmail.vanderbilt.edu (David.McFarland@mcmail.vanderbilt.edu)
Date: Thu, 6 Apr 2000 15:23:54 -0500
Subject: Cytometry journal contact info
Message-ID: <862568B9.006F9ABB.00@MCSMTP.MC.VANDERBILT.EDU>




I'm having trouble contacting the publishers of Cytometry. I'm about to
relocate and I need to make sure that my journal subscription follows me. I get
no response from the email address given as a contact at the Wiley Interscience
website. Could someone provide me with an active email address for
wiley/cytometry customer support? Thanks.

David McFarland
Howard Hughes Medical Institute
Flow Cytometry Facility
Vanderbilt University Medical Center




From A.Smith at centenary.usyd.edu.AU Thu Apr 6 18:04:46 2000
From: A.Smith at centenary.usyd.edu.AU (Adrian Smith)
Date: Fri, 7 Apr 2000 09:04:46 +1000
Subject: Applescript and Flowjo?
In-Reply-To: <p04310102b5116dd7c984@[192.94.249.197]>
References: <p04310102b5116dd7c984@[192.94.249.197]>
Message-ID: <p04310102b512bccc21f8@[10.0.2.45]>


> Dear Flowers,
>
>Has anybody succeeded in scripting Flowjo using Applescript? It would
>be great if that were possible!!
>
>Kind regards,
>
>Jan
>--
>

There appears to be a small AppleScript dictionary for FlowJo 3.1 but
it looks to be just the standard commands, ie nothing specific to
FlowJo. Not knowing anything about programming in Apple Script I have
no idea if this is of any use but you might be able to do something
with them...

What sort of things did you want to do with AppleScript? Personally I
can't really thing anything that I do that AppleScript would make
easier/quicker, but that may be just because I don't know much about
AppleScript.

Depending on what you want to do you might be able to do it with one
of the macro programs like OneClick, Quickeys or Key Quencer. A while
ago I made a simple OneClick palette to make some FlowJo features
more accessible. It saved me a bit of time for a while but I
eventually gave up using it because OneClick seemed to decrease
FlowJo's stability.

Adrian


From s9803537 at pop3.unsw.edu.au Thu Apr 6 17:33:06 2000
From: s9803537 at pop3.unsw.edu.au (R.Wadley)
Date: Fri, 07 Apr 2000 08:33:06 +1000
Subject: cell line
Message-ID: <3.0.1.32.20000407083306.007a81b0@pop3.unsw.edu.au>


Hi Flowers

Just passing this on.

Regards

Rob W.

I am looking for a cell line of mouse origin that produces IgE directed
against DNP. Our previous hybridoma (H1E-026) was sadly lost in a liquid
nitrogen mishap, and I am having trouble sourcing a replacement.
any help would be great, thanks

~~~~~~~~~~~~~~~~~~********************************************~~~~~~~~~~~~~~~~

neralie coulston
school of microbiology and immunology
University of New South Wales
Sydney, Australia
ph 9385 1081
fax 9385 1591

******************~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~*************
***






R. Wadley, B.App.Sc. M.L.S, Grad.Dip.Sc.MM
Laboratory Manager
Cellular Analysis Facility
School of Microbiology & Immunology
UNSW, New South Wales, Australia, 2052
Ph (BH) +61 (2) 9385 3517
Ph (AH) +61 (2) 9555 1239
Fax +61 (2) 9385 1591
E-mail r.wadley at unsw.edu.au
www http://www.micro.unsw.edu.au/caf.html


From Alice.L.Givan at Dartmouth.EDU Thu Apr 6 14:20:56 2000
From: Alice.L.Givan at Dartmouth.EDU (Alice L. Givan)
Date: 06 Apr 2000 15:20:56 EDT
Subject: sample/sheath ratio after sort
Message-ID: <9479722@cupid.Dartmouth.EDU>

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From justin-fishbaugh at uiowa.edu Thu Apr 6 15:52:16 2000
From: justin-fishbaugh at uiowa.edu (Justin Fishbaugh)
Date: Thu, 06 Apr 2000 15:52:16 -0500
Subject: DVD-RAM for data storage
Message-ID: <3.0.32.20000406155212.006e9590@flocyt.int-med.uiowa.edu>


Greetings,

We are in the process of switching to a larger and faster data server
(Windows NT) to store our flow cytometry data. We will be using either one
or two 9GB drives. I am wrestling with long term archive options that
currently include 4mm DAT tape, CD-RW/CD-R or DVD-RAM. I am leaning toward
DVD-RAM because of the 5.2GB capacity and longevity of the media. The down
side is that DVD-RAM is not a mature technology like CD-RW/CD-R or 4mm DAT.
We'll probably install it on a fast MAC in the lab (the server is in
another building) and use Retrospect backup software so that we can retain
Cellquest file attributes without Filetyping when we pull archived files
from the disk. The last threads in the flow e-mail archives relating to
DVD are dated 1997. Has anyone used this storage technology since then?
Thanks.




------------------------------------------------
| Justin Fishbaugh |
| University of Iowa |
| Flow Cytometry Facility |
| 48 EMRB |
| Iowa City, IA 52242 |
| |
| justin-fishbaugh at uiowa.edu |
| http://www.medicine.uiowa.edu/flowcytometry/ |
------------------------------------------------


From c.mccowan at pgrad.unimelb.edu.au Thu Apr 6 18:43:06 2000
From: c.mccowan at pgrad.unimelb.edu.au (Christina McCowan)
Date: Fri, 07 Apr 2000 09:43:06 +1000
Subject: elastase in neurtophils
In-Reply-To: <01bf9f59$a6b74e80$2e96a5d8@triss2>
Message-ID: <3.0.1.32.20000407094306.006b8860@mail.student.unimelb.edu.au>


Virginia,

You might like also to check out the Cell Probe reagents marketed by
Beckman Coulter.

http://134.217.3.35/coulter/cytometry/CellProbe-Reagents/cp-index.asp


Christina McCowan


At 18:49 05-04-2000 -0500, you wrote:
>
>Virginia,
>
>I have not tried this; but, you micht consider looking at this possibility.
>http://www.phiphilux.com/elas1.htm
>I think this is suppose to be a cell permeable substrate that changes
>fluorescent properties upon protease cleavage.
>Ty Lee
>
>
>-----Original Message-----
>From: Virginia M Litwin <virginia.litwin at bms.com>
>To: cyto-inbox
>Date: Wednesday, April 05, 2000 5:12 PM
>Subject: elastase in neurtophils
>
>
>>Does anyone know of a method to measure elastase in neutorphils?
>>
>


From adam at treestar.com Thu Apr 6 19:19:50 2000
From: adam at treestar.com (Adam Treister)
Date: Thu, 06 Apr 2000 17:19:50 -0700
Subject: Applescript and Flowjo?
Message-ID: <Mailstrom_v2.0.6.30646.4294964182.treestar@smtp.professionals.com>


----->>> On 6-Apr-2000, Jan Hendrikx wrote:
> Has anybody succeeded in scripting Flowjo using Applescript?
> It would be great if that were possible!!
--------------
I know I haven't.

As Adrian noted, FlowJo does support some minimal level of AppleScript, but
doesn't have the object model implementation which is what you'd need to do
anything meaningful. I did this in an earlier application (FlexiTrace) and
it turned into a huge amount of work to enable obtuse functionality to a
small number of users. My personal experience with AppleScripts is that
they're really brittle. If you move something, or want to something
different from the script writers intention, the script gets annoyingly
confused and its much easier to start over than to fix it. Publish and
Subscribe is an easier addition feature we've considered, but there just
aren't many people who'd take advantage of this if it were included. (Let
me know if you think otherwise.)

If properly constructed, a workspace in FlowJo is the script of all
analyses and reports in that experiment. Dropping your new data folder
onto the workspace should perform all the analyses that one might automate
with AppleScript. We have considered automating it further, but the
logical next step is auto-printing. In deference to trees, we chose to
stop short of automating the output without making you look at the data.

Someday soon, FlowJo will store its workspace in an XML syntax, which means
that we can utililze generic scripting tools, or you can even hack the
files yourself. I think this is a better approach than addiing
AppleScript.

Adam


----------------------------------
Adam Treister
Tree Star, Inc.
ph: 650-508-9349
fax: 650-508-9186
www.treestar.com
----------------------------------





From devans at calc.vet.uga.edu Fri Apr 7 11:00:37 2000
From: devans at calc.vet.uga.edu (Donald Evans)
Date: Fri, 7 Apr 2000 11:00:37 EST
Subject: Postdoctoral position
Message-ID: <200004071500.KAA11108@flowcyt.cyto.purdue.edu>


Hey:



A postdoctoral position is available at the University of Georgia
College of Veterinary Medicine. The project concerns molecular
immunology level studies of signaling in Teleost nonspecific
cytotoxic cells. A strong interest in molecular biology and flow
cytometry is desireable. Send statement of interest, curriculum
vitae and addresses of three references to: Dr. L. Jaso-Friedmann,
Department of Medical Microbiology and Parasitology, College of
Veterinary Medicine, Athens, GA. 30602.


From Altterrain at AOL.COM Fri Apr 7 14:31:52 2000
From: Altterrain at AOL.COM (Altterrain@AOL.COM)
Date: Fri, 7 Apr 2000 15:31:52 EDT
Subject: Is there a difference between CD34 and CD34+?
Message-ID: <ab.1f47991.261f91a8@aol.com>


Eileen and all,

The "+" in CD34+ simply refers to being positive for the CD34 cell surface marker. The
absence of the "+" in CD34 lymphocytes is just shorthand (or laziness).

-Brian White
Human Genome Sciences
Rockville, MD
brian_white at hgsi.com

I am a data manager for a pharmaceutical company and have the simple
question what is the meaning of the + in CD34+? and is CD34 lymphocytes the
same as CD34+ lymphocytes?

Many thanks,

> Eileen Gardenhire
> Project Data Manager
> Aventis
> PO Box 6800
> Rt. 202-206
> Bridgewater, NJ 08807-0800
> email: eileen.gardenhire at aventis.com
> tele.: 908-231-2464
> fax: 908-231-2136


From bone.bradley at MAYO.EDU Fri Apr 7 14:10:07 2000
From: bone.bradley at MAYO.EDU (Bone, Bradley G.)
Date: Fri, 7 Apr 2000 12:10:07 -0700
Subject: anti-hamster ab's
Message-ID: <A7B56CCD1E1DD211853D00A0C9A35345028FC3C3@excsrv05.mayo.edu>


Try Jackson Immunoresearch. They have a lot of secondary antibodies against
a wide range of species. I am currently using their hamster antibody in my
ELISA applications. They have antibody available against either Syrian or
Armenian Hamsters.

Regards,
Bradley
~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-

Bradley Bone
Immunology Core Facility Coordinator
Samuel C. Johnson Research Center
Mayo Clinic Scottsdale
13400 East Shea Boulevard
Scottsdale, Arizona 85259

Phone: (480) 301-7131
Pager: (480) 301-5023


-----Original Message-----
From: rose_hughes at hgsi.com [mailto:rose_hughes at hgsi.com]
Sent: Thursday, April 06, 2000 1:04 PM
To: cyto-inbox
Subject: anti-hamster ab's



Dear Flowers,

I am inquiring about a source of anti-hamster antibodies. Any leads will
be appreciated.

Rose Hughes, B.S. CT(ASCP),(IAC),CLsp(CG)
Flow Cytometry Lab
Human Genome Sciences, Inc.
9410 Key West Ave.
Rockville, MD 20850
(301) 309-8504 ext. 2074



From simmmmer at yahoo.com Fri Apr 7 16:33:21 2000
From: simmmmer at yahoo.com (Maciej Simm)
Date: Fri, 7 Apr 2000 14:33:21 -0700 (PDT)
Subject: DVD-RAM for data storage
Message-ID: <20000407213321.27465.qmail@web2004.mail.yahoo.com>


You didn't specify how much data your lab generates, however your
point about immaturity of the technology is valid.

My vote is still for cdr/w due to ready availability and
intercomputer compatibility (the dvdram media aren't compatible with
many computers at this point).

Also there is a huge price difference. A decent CDRW drive costs a
fraction of what a dvd ram drive would.

Further if you decide to invest into a cdrw drive, I recommend
againts external drives (network your computers) and between IDE and
SCSI I would definitely go SCSI for increased
performance/reliability. Also I would not settle for no-brand media.

I would also like to point out, getting back to data volume, that new
CD media are sized at 700 megs now and these discs are compatible
with older (within reason) drives. It would take 10 minutes to
generate one of these discs with an 8X burner (plus or minus
initiation/finalization of the disc) and as far as media endurance, I
have yet to find one CD that has failed me. Also due to the fact that
its a fast and cheap technology there is nothing preventing you to
get dual jewel cases and automatically burn 2 copies at each session!

=====
`---------------------------------------------`
| Maciej S. Simm | 525 E 68th Street |
| Research Technician | Room N-805 |
| Cornell Medical Center | Tel. 212.746.3428 |
`---------------------------------------------`
| www.cd4cd8.com |
`---------------------------------------------`

__________________________________________________
Do You Yahoo!?
Talk to your friends online with Yahoo! Messenger.
http://im.yahoo.com


From Oconnell at Moffitt.usf.edu Fri Apr 7 10:26:17 2000
From: Oconnell at Moffitt.usf.edu (O'Connell, Christine)
Date: Fri, 7 Apr 2000 11:26:17 -0400
Subject: Job Opportunity - Florida
Message-ID: <37B672BBC0D3D111AD6900805FE61E700457377C@hlmex02.moffitt.usf.edu>


H. Lee Moffitt Cancer Center & Research Institute
April 7, 2000
The Flow Cytometry Core Facility is currently seeking to fill the following
position:
Staff Scientist
The Flow Cytometry Core Facility at H. Lee Moffitt Cancer Center & Research
Institute, located on the University of South Florida Campus, has a
Non-Faculty - STAFF SCIENTIST position available in the Flow Cytometry Core
Facility.
The Staff Scientist is responsible for providing technical expertise,
advice, and oversight of ongoing and future projects that utilize the
facility. The Core is seeking a highly motivated individual with the
following skills:
Education: Minimum of Masters Degree in the Biological Science or related
field with appropriate flow cytometry training (with at least 3-5 years of
HANDS-ON sorting experience) or a Bachelors Degree with extensive HANDS-ON
experience.
* Hands-on experience with research flow cytometers (sorters such as
the FACStar, FACStar plus, Vantage, Vantage SE, or MoFlo).
* Flow cytometry techniques such as multi-parameter sorting, high
speed sorting, data acquisition and analysis.
* Experience with cell cycle acquisition and analysis.
* Excellent written and verbal communication skills are required.
* The Core Facility has the following instrumentation:
* FACStar Plus (2 laser bench)
* Vantage SE (turbo sorter, 3-laser bench)
* 2 - FACScans
* Software: CellQuest, MODFit, FLOJo
Moffitt offers an excellent salary/benefits program and professional
academic environment. Applications will be reviewed immediately until the
position is filled.


To apply, send/fax resume or CV to:
Human Resources
MOFFITT CANCER CENTER
12902 Magnolia Drive, Tampa Florida 33612
FAX: 813-979-6700
OR e-mail to: oconnell at moffitt.usf.edu <mailto:oconnell at moffitt.usf.edu>
Moffitt Cancer Center is a Drug Free Workplace.
Affirmative Action Employer.

Christine O'Connell
Director, Research Laboratory Operations
Moffitt Cancer Center
813-632-1357 (phone)
813-979-6700 (fax)
oconnell at moffitt.usf.edu <mailto:oconnell at moffitt.usf.edu>



From thomas at tritechinc.com Fri Apr 7 15:28:14 2000
From: thomas at tritechinc.com (Joanne Thomas)
Date: Fri, 7 Apr 2000 16:28:14 -0400
Subject: Cellquest Availability
Message-ID: <002d01bfa0cf$cf2758c0$1801a8c0@jill.tritechinc.com>

Hi All!
We have been trying to purchase the new version of CellQuest which is supposed to address the OS 9 issues and have been told that it is on back order for some time now. Has anyone else had a problem getting version 3.3???

Joanne Thomas, M.S.
Director of Operations
TRITECH Field Engineering
2014 Renard Court, Suite I
Annapolis, MD 21401
1-800-886-7004 (USA)
1-410-266-1522
410-266-0935 (FAX)
thomas at tritechinc.com
-------------- next part --------------
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From jpr at flowcyt.cyto.purdue.edu Fri Apr 7 17:22:54 2000
From: jpr at flowcyt.cyto.purdue.edu (jpr@flowcyt.cyto.purdue.edu)
Date: Fri, 7 Apr 2000 17:22:54 -0500
Subject: The last mesage on Cytometry CD5
Message-ID: <38EE196E.9027.4105E5@localhost>


Dear Colleagues:
This is the last message that will appear on the cytometry discussion
on the 5th volume of our Cytometry CD-ROM series from Purdue
University.

Thanks to all those who contributed. I am excited about this CD-
ROM. For a sneak preview go to
www.cyto.purdue.edu/flowcyt/cdseries.htm

Through a collaboration with ISAC and Wiley-Liss, we have been
able to provide all of the ISAC XX abstracts, including the tables and
figures that some of you placed in your abstracts. Some people
provided a considerable amount of additional information to
complement their abstracts. The CDROM was sponsored by 14
companies, most of which will have booths at the ISAC congress.
You will be able to obtain your free copy from one of these
sponsors. Included in your ISAC Congress packets will be a ticket
that can be redeemed for a free CD-ROM from any one of the 14
sponsors at their booths. The sponsors were Dako, Bangs Labs, IQ
Products, BD Biosciences, Beckman-Coulter, Diatec, Caltag,
Treestar, Cytomation, CLB, Tritech, BioE, Phoenix Flow, and
Southern Biotechnology Associates. Please search out these
sponsors who paid the costs of the CD-ROM so that it can be
distributed free at the congress. They are also all good supporters of
our ISAC congress of course.

Apart from the Abstracts, there is a lot of useful information on CD5 -
Powerpoint lectures, image sets, data sets, some useful protocols,
clinical and research materials and lots of other educational
material.

The CDs can be used on any WEB browser (and yes this one was
tested on the MAC!!) and works on or off the internet. The staff of
Purdue University Cytometry Labs, have worked hard to make this
the best CD yet - Monica Shively and her team have done a nice job
for which I am very grateful.

Look for the CD at ISAC - don't forget to pick up a copy, and if you
have some material you think should be on the next one - send it to
us.

So, until Montpellier then.....
Best regards

Paul Robinson, CD5 Publisher
Monica Shively, Project Director


J.Paul Robinson, PhD PH:(765)4940757
Professor of Immunopharmacology
Professor of Biomedical Engineering
Purdue University FAX:(765)4940517
EMAIL:jpr at flowcyt.cyto.purdue.edu
WEB: http://www.cyto.purdue.edu


From Eileen.Gardenhire at aventis.com Thu Apr 6 17:39:11 2000
From: Eileen.Gardenhire at aventis.com (Eileen.Gardenhire@aventis.com)
Date: Thu, 6 Apr 2000 17:39:11 -0500
Subject: Is there a difference between CD34 and CD34+?
Message-ID: <AFF2E1D861D8D111A34100A0C9AF9909015760F8@brwsmxsusr07.brw.hmrag.com>


I am a data manager for a pharmaceutical company and have the simple
question what is the meaning of the + in CD34+? and is CD34 lymphocytes the
same as CD34+ lymphocytes?

Many thanks,

> Eileen Gardenhire
> Project Data Manager
> Aventis
> PO Box 6800
> Rt. 202-206
> Bridgewater, NJ 08807-0800
> email: eileen.gardenhire at aventis.com
> tele.: 908-231-2464
> fax: 908-231-2136
>
>


From rose_hughes at hgsi.com Thu Apr 6 15:04:26 2000
From: rose_hughes at hgsi.com (rose_hughes@hgsi.com)
Date: Thu, 6 Apr 2000 16:04:26 -0400
Subject: anti-hamster ab's
Message-ID: <OFD9F48B9F.A1D86350-ON852568B9.006DE7BF@hgsi.com>


Dear Flowers,

I am inquiring about a source of anti-hamster antibodies. Any leads will
be appreciated.

Rose Hughes, B.S. CT(ASCP),(IAC),CLsp(CG)
Flow Cytometry Lab
Human Genome Sciences, Inc.
9410 Key West Ave.
Rockville, MD 20850
(301) 309-8504 ext. 2074




From David.McFarland at mcmail.vanderbilt.edu Thu Apr 6 15:23:54 2000
From: David.McFarland at mcmail.vanderbilt.edu (David.McFarland@mcmail.vanderbilt.edu)
Date: Thu, 6 Apr 2000 15:23:54 -0500
Subject: Cytometry journal contact info
Message-ID: <862568B9.006F9ABB.00@MCSMTP.MC.VANDERBILT.EDU>




I'm having trouble contacting the publishers of Cytometry. I'm about to
relocate and I need to make sure that my journal subscription follows me. I get
no response from the email address given as a contact at the Wiley Interscience
website. Could someone provide me with an active email address for
wiley/cytometry customer support? Thanks.

David McFarland
Howard Hughes Medical Institute
Flow Cytometry Facility
Vanderbilt University Medical Center




From A.Smith at centenary.usyd.edu.AU Thu Apr 6 18:04:46 2000
From: A.Smith at centenary.usyd.edu.AU (Adrian Smith)
Date: Fri, 7 Apr 2000 09:04:46 +1000
Subject: Applescript and Flowjo?
In-Reply-To: <p04310102b5116dd7c984@[192.94.249.197]>
References: <p04310102b5116dd7c984@[192.94.249.197]>
Message-ID: <p04310102b512bccc21f8@[10.0.2.45]>


> Dear Flowers,
>
>Has anybody succeeded in scripting Flowjo using Applescript? It would
>be great if that were possible!!
>
>Kind regards,
>
>Jan
>--
>

There appears to be a small AppleScript dictionary for FlowJo 3.1 but
it looks to be just the standard commands, ie nothing specific to
FlowJo. Not knowing anything about programming in Apple Script I have
no idea if this is of any use but you might be able to do something
with them...

What sort of things did you want to do with AppleScript? Personally I
can't really thing anything that I do that AppleScript would make
easier/quicker, but that may be just because I don't know much about
AppleScript.

Depending on what you want to do you might be able to do it with one
of the macro programs like OneClick, Quickeys or Key Quencer. A while
ago I made a simple OneClick palette to make some FlowJo features
more accessible. It saved me a bit of time for a while but I
eventually gave up using it because OneClick seemed to decrease
FlowJo's stability.

Adrian


From s9803537 at pop3.unsw.edu.au Thu Apr 6 17:33:06 2000
From: s9803537 at pop3.unsw.edu.au (R.Wadley)
Date: Fri, 07 Apr 2000 08:33:06 +1000
Subject: cell line
Message-ID: <3.0.1.32.20000407083306.007a81b0@pop3.unsw.edu.au>


Hi Flowers

Just passing this on.

Regards

Rob W.

I am looking for a cell line of mouse origin that produces IgE directed
against DNP. Our previous hybridoma (H1E-026) was sadly lost in a liquid
nitrogen mishap, and I am having trouble sourcing a replacement.
any help would be great, thanks

~~~~~~~~~~~~~~~~~~********************************************~~~~~~~~~~~~~~~~

neralie coulston
school of microbiology and immunology
University of New South Wales
Sydney, Australia
ph 9385 1081
fax 9385 1591

******************~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~*************
***






R. Wadley, B.App.Sc. M.L.S, Grad.Dip.Sc.MM
Laboratory Manager
Cellular Analysis Facility
School of Microbiology & Immunology
UNSW, New South Wales, Australia, 2052
Ph (BH) +61 (2) 9385 3517
Ph (AH) +61 (2) 9555 1239
Fax +61 (2) 9385 1591
E-mail r.wadley at unsw.edu.au
www http://www.micro.unsw.edu.au/caf.html


From Alice.L.Givan at Dartmouth.EDU Thu Apr 6 14:20:56 2000
From: Alice.L.Givan at Dartmouth.EDU (Alice L. Givan)
Date: 06 Apr 2000 15:20:56 EDT
Subject: sample/sheath ratio after sort
Message-ID: <9479722@cupid.Dartmouth.EDU>

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From justin-fishbaugh at uiowa.edu Thu Apr 6 15:52:16 2000
From: justin-fishbaugh at uiowa.edu (Justin Fishbaugh)
Date: Thu, 06 Apr 2000 15:52:16 -0500
Subject: DVD-RAM for data storage
Message-ID: <3.0.32.20000406155212.006e9590@flocyt.int-med.uiowa.edu>


Greetings,

We are in the process of switching to a larger and faster data server
(Windows NT) to store our flow cytometry data. We will be using either one
or two 9GB drives. I am wrestling with long term archive options that
currently include 4mm DAT tape, CD-RW/CD-R or DVD-RAM. I am leaning toward
DVD-RAM because of the 5.2GB capacity and longevity of the media. The down
side is that DVD-RAM is not a mature technology like CD-RW/CD-R or 4mm DAT.
We'll probably install it on a fast MAC in the lab (the server is in
another building) and use Retrospect backup software so that we can retain
Cellquest file attributes without Filetyping when we pull archived files
from the disk. The last threads in the flow e-mail archives relating to
DVD are dated 1997. Has anyone used this storage technology since then?
Thanks.




------------------------------------------------
| Justin Fishbaugh |
| University of Iowa |
| Flow Cytometry Facility |
| 48 EMRB |
| Iowa City, IA 52242 |
| |
| justin-fishbaugh at uiowa.edu |
| http://www.medicine.uiowa.edu/flowcytometry/ |
------------------------------------------------


From c.mccowan at pgrad.unimelb.edu.au Thu Apr 6 18:43:06 2000
From: c.mccowan at pgrad.unimelb.edu.au (Christina McCowan)
Date: Fri, 07 Apr 2000 09:43:06 +1000
Subject: elastase in neurtophils
In-Reply-To: <01bf9f59$a6b74e80$2e96a5d8@triss2>
Message-ID: <3.0.1.32.20000407094306.006b8860@mail.student.unimelb.edu.au>


Virginia,

You might like also to check out the Cell Probe reagents marketed by
Beckman Coulter.

http://134.217.3.35/coulter/cytometry/CellProbe-Reagents/cp-index.asp


Christina McCowan


At 18:49 05-04-2000 -0500, you wrote:
>
>Virginia,
>
>I have not tried this; but, you micht consider looking at this possibility.
>http://www.phiphilux.com/elas1.htm
>I think this is suppose to be a cell permeable substrate that changes
>fluorescent properties upon protease cleavage.
>Ty Lee
>
>
>-----Original Message-----
>From: Virginia M Litwin <virginia.litwin at bms.com>
>To: cyto-inbox
>Date: Wednesday, April 05, 2000 5:12 PM
>Subject: elastase in neurtophils
>
>
>>Does anyone know of a method to measure elastase in neutorphils?
>>
>


From adam at treestar.com Thu Apr 6 19:19:50 2000
From: adam at treestar.com (Adam Treister)
Date: Thu, 06 Apr 2000 17:19:50 -0700
Subject: Applescript and Flowjo?
Message-ID: <Mailstrom_v2.0.6.30646.4294964182.treestar@smtp.professionals.com>


----->>> On 6-Apr-2000, Jan Hendrikx wrote:
> Has anybody succeeded in scripting Flowjo using Applescript?
> It would be great if that were possible!!
--------------
I know I haven't.

As Adrian noted, FlowJo does support some minimal level of AppleScript, but
doesn't have the object model implementation which is what you'd need to do
anything meaningful. I did this in an earlier application (FlexiTrace) and
it turned into a huge amount of work to enable obtuse functionality to a
small number of users. My personal experience with AppleScripts is that
they're really brittle. If you move something, or want to something
different from the script writers intention, the script gets annoyingly
confused and its much easier to start over than to fix it. Publish and
Subscribe is an easier addition feature we've considered, but there just
aren't many people who'd take advantage of this if it were included. (Let
me know if you think otherwise.)

If properly constructed, a workspace in FlowJo is the script of all
analyses and reports in that experiment. Dropping your new data folder
onto the workspace should perform all the analyses that one might automate
with AppleScript. We have considered automating it further, but the
logical next step is auto-printing. In deference to trees, we chose to
stop short of automating the output without making you look at the data.

Someday soon, FlowJo will store its workspace in an XML syntax, which means
that we can utililze generic scripting tools, or you can even hack the
files yourself. I think this is a better approach than addiing
AppleScript.

Adam


----------------------------------
Adam Treister
Tree Star, Inc.
ph: 650-508-9349
fax: 650-508-9186
www.treestar.com
----------------------------------





From devans at calc.vet.uga.edu Fri Apr 7 11:00:37 2000
From: devans at calc.vet.uga.edu (Donald Evans)
Date: Fri, 7 Apr 2000 11:00:37 EST
Subject: Postdoctoral position
Message-ID: <200004071500.KAA11108@flowcyt.cyto.purdue.edu>


Hey:



A postdoctoral position is available at the University of Georgia
College of Veterinary Medicine. The project concerns molecular
immunology level studies of signaling in Teleost nonspecific
cytotoxic cells. A strong interest in molecular biology and flow
cytometry is desireable. Send statement of interest, curriculum
vitae and addresses of three references to: Dr. L. Jaso-Friedmann,
Department of Medical Microbiology and Parasitology, College of
Veterinary Medicine, Athens, GA. 30602.


From Altterrain at AOL.COM Fri Apr 7 14:31:52 2000
From: Altterrain at AOL.COM (Altterrain@AOL.COM)
Date: Fri, 7 Apr 2000 15:31:52 EDT
Subject: Is there a difference between CD34 and CD34+?
Message-ID: <ab.1f47991.261f91a8@aol.com>


Eileen and all,

The "+" in CD34+ simply refers to being positive for the CD34 cell surface marker. The
absence of the "+" in CD34 lymphocytes is just shorthand (or laziness).

-Brian White
Human Genome Sciences
Rockville, MD
brian_white at hgsi.com

I am a data manager for a pharmaceutical company and have the simple
question what is the meaning of the + in CD34+? and is CD34 lymphocytes the
same as CD34+ lymphocytes?

Many thanks,

> Eileen Gardenhire
> Project Data Manager
> Aventis
> PO Box 6800
> Rt. 202-206
> Bridgewater, NJ 08807-0800
> email: eileen.gardenhire at aventis.com
> tele.: 908-231-2464
> fax: 908-231-2136


From bone.bradley at MAYO.EDU Fri Apr 7 14:10:07 2000
From: bone.bradley at MAYO.EDU (Bone, Bradley G.)
Date: Fri, 7 Apr 2000 12:10:07 -0700
Subject: anti-hamster ab's
Message-ID: <A7B56CCD1E1DD211853D00A0C9A35345028FC3C3@excsrv05.mayo.edu>


Try Jackson Immunoresearch. They have a lot of secondary antibodies against
a wide range of species. I am currently using their hamster antibody in my
ELISA applications. They have antibody available against either Syrian or
Armenian Hamsters.

Regards,
Bradley
~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-~-

Bradley Bone
Immunology Core Facility Coordinator
Samuel C. Johnson Research Center
Mayo Clinic Scottsdale
13400 East Shea Boulevard
Scottsdale, Arizona 85259

Phone: (480) 301-7131
Pager: (480) 301-5023


-----Original Message-----
From: rose_hughes at hgsi.com [mailto:rose_hughes at hgsi.com]
Sent: Thursday, April 06, 2000 1:04 PM
To: cyto-inbox
Subject: anti-hamster ab's



Dear Flowers,

I am inquiring about a source of anti-hamster antibodies. Any leads will
be appreciated.

Rose Hughes, B.S. CT(ASCP),(IAC),CLsp(CG)
Flow Cytometry Lab
Human Genome Sciences, Inc.
9410 Key West Ave.
Rockville, MD 20850
(301) 309-8504 ext. 2074



From simmmmer at yahoo.com Fri Apr 7 16:33:21 2000
From: simmmmer at yahoo.com (Maciej Simm)
Date: Fri, 7 Apr 2000 14:33:21 -0700 (PDT)
Subject: DVD-RAM for data storage
Message-ID: <20000407213321.27465.qmail@web2004.mail.yahoo.com>


You didn't specify how much data your lab generates, however your
point about immaturity of the technology is valid.

My vote is still for cdr/w due to ready availability and
intercomputer compatibility (the dvdram media aren't compatible with
many computers at this point).

Also there is a huge price difference. A decent CDRW drive costs a
fraction of what a dvd ram drive would.

Further if you decide to invest into a cdrw drive, I recommend
againts external drives (network your computers) and between IDE and
SCSI I would definitely go SCSI for increased
performance/reliability. Also I would not settle for no-brand media.

I would also like to point out, getting back to data volume, that new
CD media are sized at 700 megs now and these discs are compatible
with older (within reason) drives. It would take 10 minutes to
generate one of these discs with an 8X burner (plus or minus
initiation/finalization of the disc) and as far as media endurance, I
have yet to find one CD that has failed me. Also due to the fact that
its a fast and cheap technology there is nothing preventing you to
get dual jewel cases and automatically burn 2 copies at each session!

=====
`---------------------------------------------`
| Maciej S. Simm | 525 E 68th Street |
| Research Technician | Room N-805 |
| Cornell Medical Center | Tel. 212.746.3428 |
`---------------------------------------------`
| www.cd4cd8.com |
`---------------------------------------------`

__________________________________________________
Do You Yahoo!?
Talk to your friends online with Yahoo! Messenger.
http://im.yahoo.com


From Oconnell at Moffitt.usf.edu Fri Apr 7 10:26:17 2000
From: Oconnell at Moffitt.usf.edu (O'Connell, Christine)
Date: Fri, 7 Apr 2000 11:26:17 -0400
Subject: Job Opportunity - Florida
Message-ID: <37B672BBC0D3D111AD6900805FE61E700457377C@hlmex02.moffitt.usf.edu>


H. Lee Moffitt Cancer Center & Research Institute
April 7, 2000
The Flow Cytometry Core Facility is currently seeking to fill the following
position:
Staff Scientist
The Flow Cytometry Core Facility at H. Lee Moffitt Cancer Center & Research
Institute, located on the University of South Florida Campus, has a
Non-Faculty - STAFF SCIENTIST position available in the Flow Cytometry Core
Facility.
The Staff Scientist is responsible for providing technical expertise,
advice, and oversight of ongoing and future projects that utilize the
facility. The Core is seeking a highly motivated individual with the
following skills:
Education: Minimum of Masters Degree in the Biological Science or related
field with appropriate flow cytometry training (with at least 3-5 years of
HANDS-ON sorting experience) or a Bachelors Degree with extensive HANDS-ON
experience.
* Hands-on experience with research flow cytometers (sorters such as
the FACStar, FACStar plus, Vantage, Vantage SE, or MoFlo).
* Flow cytometry techniques such as multi-parameter sorting, high
speed sorting, data acquisition and analysis.
* Experience with cell cycle acquisition and analysis.
* Excellent written and verbal communication skills are required.
* The Core Facility has the following instrumentation:
* FACStar Plus (2 laser bench)
* Vantage SE (turbo sorter, 3-laser bench)
* 2 - FACScans
* Software: CellQuest, MODFit, FLOJo
Moffitt offers an excellent salary/benefits program and professional
academic environment. Applications will be reviewed immediately until the
position is filled.


To apply, send/fax resume or CV to:
Human Resources
MOFFITT CANCER CENTER
12902 Magnolia Drive, Tampa Florida 33612
FAX: 813-979-6700
OR e-mail to: oconnell at moffitt.usf.edu <mailto:oconnell at moffitt.usf.edu>
Moffitt Cancer Center is a Drug Free Workplace.
Affirmative Action Employer.

Christine O'Connell
Director, Research Laboratory Operations
Moffitt Cancer Center
813-632-1357 (phone)
813-979-6700 (fax)
oconnell at moffitt.usf.edu <mailto:oconnell at moffitt.usf.edu>



From thomas at tritechinc.com Fri Apr 7 15:28:14 2000
From: thomas at tritechinc.com (Joanne Thomas)
Date: Fri, 7 Apr 2000 16:28:14 -0400
Subject: Cellquest Availability
Message-ID: <002d01bfa0cf$cf2758c0$1801a8c0@jill.tritechinc.com>

Hi All!
We have been trying to purchase the new version of CellQuest which is supposed to address the OS 9 issues and have been told that it is on back order for some time now. Has anyone else had a problem getting version 3.3???

Joanne Thomas, M.S.
Director of Operations
TRITECH Field Engineering
2014 Renard Court, Suite I
Annapolis, MD 21401
1-800-886-7004 (USA)
1-410-266-1522
410-266-0935 (FAX)
thomas at tritechinc.com
-------------- next part --------------
HTML attachment scrubbed and removed

From jpr at flowcyt.cyto.purdue.edu Fri Apr 7 17:22:54 2000
From: jpr at flowcyt.cyto.purdue.edu (jpr@flowcyt.cyto.purdue.edu)
Date: Fri, 7 Apr 2000 17:22:54 -0500
Subject: The last mesage on Cytometry CD5
Message-ID: <38EE196E.9027.4105E5@localhost>


Dear Colleagues:
This is the last message that will appear on the cytometry discussion
on the 5th volume of our Cytometry CD-ROM series from Purdue
University.

Thanks to all those who contributed. I am excited about this CD-
ROM. For a sneak preview go to
www.cyto.purdue.edu/flowcyt/cdseries.htm

Through a collaboration with ISAC and Wiley-Liss, we have been
able to provide all of the ISAC XX abstracts, including the tables and
figures that some of you placed in your abstracts. Some people
provided a considerable amount of additional information to
complement their abstracts. The CDROM was sponsored by 14
companies, most of which will have booths at the ISAC congress.
You will be able to obtain your free copy from one of these
sponsors. Included in your ISAC Congress packets will be a ticket
that can be redeemed for a free CD-ROM from any one of the 14
sponsors at their booths. The sponsors were Dako, Bangs Labs, IQ
Products, BD Biosciences, Beckman-Coulter, Diatec, Caltag,
Treestar, Cytomation, CLB, Tritech, BioE, Phoenix Flow, and
Southern Biotechnology Associates. Please search out these
sponsors who paid the costs of the CD-ROM so that it can be
distributed free at the congress. They are also all good supporters of
our ISAC congress of course.

Apart from the Abstracts, there is a lot of useful information on CD5 -
Powerpoint lectures, image sets, data sets, some useful protocols,
clinical and research materials and lots of other educational
material.

The CDs can be used on any WEB browser (and yes this one was
tested on the MAC!!) and works on or off the internet. The staff of
Purdue University Cytometry Labs, have worked hard to make this
the best CD yet - Monica Shively and her team have done a nice job
for which I am very grateful.

Look for the CD at ISAC - don't forget to pick up a copy, and if you
have some material you think should be on the next one - send it to
us.

So, until Montpellier then.....
Best regards

Paul Robinson, CD5 Publisher
Monica Shively, Project Director


J.Paul Robinson, PhD PH:(765)4940757
Professor of Immunopharmacology
Professor of Biomedical Engineering
Purdue University FAX:(765)4940517
EMAIL:jpr at flowcyt.cyto.purdue.edu
WEB: http://www.cyto.purdue.edu


From mahmoo_a.MED.TMU at NET1CS.modares.ac.ir Mon Apr 10 12:38:38 2000
From: mahmoo_a.MED.TMU at NET1CS.modares.ac.ir (mahmoo_a.MED.TMU@NET1CS.modares.ac.ir)
Date: Mon, 10 Apr 2000 12:38:38
Subject: Ph.d position
Message-ID: <7A345472B9@net1cs.modares.ac.ir>


Hello my friends:
I am looking for Ph.d position.
I am studying Hematology in M.S position.
I am working about Differentiation therapy.
Lot thanks.
Aziz Mahmoodzadeh.
Tarbiat Modares University.
Hematolog Department.


From L.gaudry at unsw.EDU.AU Sun Apr 9 22:32:28 2000
From: L.gaudry at unsw.EDU.AU (Leonie Gaudry)
Date: Mon, 10 Apr 2000 13:32:28 +1000
Subject: Mouse Erythroid Markers
Message-ID: <3.0.6.32.20000410133228.008f5ec0@pop3.unsw.edu.au>


Hi All,
We have been using CD 71 PE to sort for erythroid precursors but are now
not sure that this is specific enough. What other erythroid markers are
available for use with mice and how specific are they?
Thank you,
Regards Leonie



From lwarma at med.unc.edu Mon Apr 10 09:54:21 2000
From: lwarma at med.unc.edu (Larry Arnold)
Date: Mon, 10 Apr 2000 09:54:21 -0500
Subject: FileGuard on MAC OS 8.6
Message-ID: <200004101352.JAA05538@zonetail.med.unc.edu>


I am about to install FileGuard on my MAC OS 8.6 platform FACSCalibur.
Does anyone have in general/specific words of wisdom?
Does it work?
Known problems?

Thanks much in advance.

Larry


Larry W. Arnold, Ph.D.
Res. Assoc. Prof.
Director, Flow Cytometry Facility
Department of Microbiology and Immunology
CB# 7290
University of North Carolina
Chapel Hill, NC 27599
Phone: 919-966-1530
FAX: 919-962-8103


From knut.egelie at diatec.com Mon Apr 10 08:29:37 2000
From: knut.egelie at diatec.com (=?iso-8859-1?Q?Knut_J=F8rgen__Egelie?=)
Date: Mon, 10 Apr 2000 15:29:37 +0200
Subject: cell line
Message-ID: <B1E9D4138E1ED311B2420060971D330E06877D@www.diatec.com>


Dear Rob

We do not produce the IgE directed against DNP, but we would like to inform
you of our product - purified monoclonal Human IgE (kappa). It is produced
in vitro by our hybridoma HE1 in accordance with ISO9001.

The original antibody producing cells were obtained from a healthy donor
tested negative for HIV, HCV and Hepatitis B using US-FDA approved tests.

The product is purified on Protein L columns and appears as a single band
after SDS-PAGE.

We supply the Human IgE in vials of 0.1 mg and 1.0 mg. We can also supply
the material in bulk quantities up to 1 gram on request.

We have available a limited number of 0.1 mg vials for testing, free of
charge.

Regards
Knut J.

Knut J. Egelie, M. Sc.,
Product Manager
DIATEC.COM: http://www.diatec.com
Tel: +47 22 95 86 25
Fax:+47 22 95 86 49


-----Original Message-----
From: R.Wadley [mailto:s9803537 at pop3.unsw.edu.au]
Sent: 6. april 2000 23:33
To: cyto-inbox
Subject: cell line



Hi Flowers

Just passing this on.

Regards

Rob W.

I am looking for a cell line of mouse origin that produces IgE directed
against DNP. Our previous hybridoma (H1E-026) was sadly lost in a liquid
nitrogen mishap, and I am having trouble sourcing a replacement.
any help would be great, thanks

~~~~~~~~~~~~~~~~~~********************************************~~~~~~~~~~~~~~
~~

neralie coulston
school of microbiology and immunology
University of New South Wales
Sydney, Australia
ph 9385 1081
fax 9385 1591

******************~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~*************
***






R. Wadley, B.App.Sc. M.L.S, Grad.Dip.Sc.MM
Laboratory Manager
Cellular Analysis Facility
School of Microbiology & Immunology
UNSW, New South Wales, Australia, 2052
Ph (BH) +61 (2) 9385 3517
Ph (AH) +61 (2) 9555 1239
Fax +61 (2) 9385 1591
E-mail r.wadley at unsw.edu.au
www http://www.micro.unsw.edu.au/caf.html


From kbahjat at ufl.edu Sat Apr 8 12:49:25 2000
From: kbahjat at ufl.edu (Keith Bahjat)
Date: Sat, 08 Apr 2000 13:49:25 -0400
Subject: Cytometry Companies and the Internet
In-Reply-To: <002d01bfa0cf$cf2758c0$1801a8c0@jill.tritechinc.com>
Message-ID: <B514E964.56B%kbahjat@ufl.edu>

Which raises an interesting point we were discussing earlier this week. As
the software that controls our cytometers, like Cellquest and EXPO, require
hardware dongles, and thus are VERY secure, why do the companies still make
us jump through hoops to get updated copies?? They could post the update on
a web site, and save the cost of having those 5000 CD's burnt every time you
upgrade to version X.X.1. The WHOLE CD with all of BD's sample files and
documentation on it is still only 40 Mb! That's not even a 7 minute download
at only 100K/sec.

Hope someone out there is listening. I think we've all got enough useless
AOL, Compuserve, and Earthlink CD's laying around. We don't need CD's from
cytometry companies on top of that. Post it on a web site.......PLEASE!

kb

Keith Bahjat
kbahjat at ufl.edu



on 4/7/00 4:28 PM, Joanne Thomas at thomas at tritechinc.com wrote:

Hi All!
We have been trying to purchase the new version of CellQuest which is
supposed to address the OS 9 issues and have been told that it is on back
order for some time now. Has anyone else had a problem getting version
3.3???

Joanne Thomas, M.S.
Director of Operations
TRITECH Field Engineering
2014 Renard Court, Suite I
Annapolis, MD 21401
1-800-886-7004 (USA)
1-410-266-1522
410-266-0935 (FAX)
thomas at tritechinc.com



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From gregor.rothe at klinik.uni-regensburg.de Mon Apr 10 02:59:05 2000
From: gregor.rothe at klinik.uni-regensburg.de (Gregor Rothe)
Date: Mon, 10 Apr 2000 09:59:05 +0200
Subject: elastase in neurtophils
In-Reply-To: <3.0.1.32.20000407094306.006b8860@mail.student.unimelb.edu.au>
Message-ID: <000401bfa2c2$a4ce1e70$22abc784@uniregensburg.de>


You should be at least extremely careful when attempting to analyse a
specific enzyme with Cell Probe reagents. In contrast to our previously
published work (Biol Chem Hoppe-Seyler 373: 547-54, 1992) on the
intracellular analysis of elastase with rhodamine 110 based substrates, many
Cell Probe reagents are not blocked against non-specific exopeptidase
activity. Also except citing the specificity of an analog non-rhodamine
based substrate in a cell lysate assay, no data are given about the
specificity of the reagents in an intracellular assay. This information
could be easily achieved using cell-permeable and specific inhibitors. I am,
however, not aware of any such data for Cell Probe reagents.

Gregor Rothe

> -----Original Message-----
> From: Christina McCowan [mailto:c.mccowan at pgrad.unimelb.edu.au]
> Sent: Friday, April 07, 2000 1:43 AM
> To: Cytometry Mailing List
> Subject: Re: elastase in neurtophils
>
>
>
> Virginia,
>
> You might like also to check out the Cell Probe reagents marketed by
> Beckman Coulter.
>
> http://134.217.3.35/coulter/cytometry/CellProbe-Reagents/cp-index.asp
>
>
> Christina McCowan
>
>
> At 18:49 05-04-2000 -0500, you wrote:
> >
> >Virginia,
> >
> >I have not tried this; but, you micht consider looking at this
> possibility.
> >http://www.phiphilux.com/elas1.htm
> >I think this is suppose to be a cell permeable substrate that changes
> >fluorescent properties upon protease cleavage.
> >Ty Lee
> >
> >
> >-----Original Message-----
> >From: Virginia M Litwin <virginia.litwin at bms.com>
> >To: cyto-inbox
> >Date: Wednesday, April 05, 2000 5:12 PM
> >Subject: elastase in neurtophils
> >
> >
> >>Does anyone know of a method to measure elastase in neutorphils?
> >>
> >
>



From tylee at itis.com Fri Apr 7 19:09:48 2000
From: tylee at itis.com (tylee)
Date: Fri, 7 Apr 2000 19:09:48 -0500
Subject: CellProbe / Re: elastase in neurtophils
Message-ID: <01bfa0ee$c16efc00$4996a5d8@triss2>


Christina,

Upon first attempt the link you provided did not work for me. I origionally
was going to respond to Virginia with information about CellProbe; but, when
I looked the other day, I found a list of about ~25-30 of Coulter's
CellProbe reagents on a DISCONTINUED PRODUCTS list.

Could a rep from Beckman/Coulter please respond to this list with an
explaination regarding why these reagents are being discontinued? Are all
of the CellProbe reagents discontinued? Is it because of lack of sales?
Does it have something to do with the Beckman takeover? I understand the
reagents have been critisized because they are susceptable to non-specific
protease (?aminopeptidase) attack. Can anyone comment about their
experience with the non-specific protease lability? Does that have anything
to do with their discontinuation?

Curious in flowland...

Ty Lee
-----Original Message-----
From: Christina McCowan <c.mccowan at pgrad.unimelb.edu.au>
To: cyto-inbox
Date: Friday, April 07, 2000 2:19 PM
Subject: Re: elastase in neurtophils


>
>Virginia,
>
>You might like also to check out the Cell Probe reagents marketed by
>Beckman Coulter.
>
>http://134.217.3.35/coulter/cytometry/CellProbe-Reagents/cp-index.asp
>
>
>Christina McCowan
>
>
>At 18:49 05-04-2000 -0500, you wrote:
>>
>>Virginia,
>>
>>I have not tried this; but, you micht consider looking at this
possibility.
>>http://www.phiphilux.com/elas1.htm
>>I think this is suppose to be a cell permeable substrate that changes
>>fluorescent properties upon protease cleavage.
>>Ty Lee
>>
>>
>>-----Original Message-----
>>From: Virginia M Litwin <virginia.litwin at bms.com>
>>To: cyto-inbox
>>Date: Wednesday, April 05, 2000 5:12 PM
>>Subject: elastase in neurtophils
>>
>>
>>>Does anyone know of a method to measure elastase in neutorphils?
>>>
>>
>



From jelewis1 at facstaff.wisc.edu Mon Apr 10 11:19:22 2000
From: jelewis1 at facstaff.wisc.edu (Janet E. Lewis)
Date: Mon, 10 Apr 2000 11:19:22 -0500
Subject: DVD-RAM for data storage
In-Reply-To: <3.0.32.20000406155212.006e9590@flocyt.int-med.uiowa.edu>
Message-ID: <4.2.2.20000410110258.01025420@facstaff.wisc.edu>


Justin,

At 03:52 PM 4/6/00 -0500, thus did you send forth:
>We are in the process of switching to a larger and faster data server
>(Windows NT) to store our flow cytometry data. We will be using either one
>or two 9GB drives. I am wrestling with long term archive options that
>currently include 4mm DAT tape, CD-RW/CD-R or DVD-RAM. I am leaning toward
>DVD-RAM because of the 5.2GB capacity and longevity of the media. The down
>side is that DVD-RAM is not a mature technology like CD-RW/CD-R or 4mm DAT.
> We'll probably install it on a fast MAC in the lab (the server is in
>another building) and use Retrospect backup software so that we can retain
>Cellquest file attributes without Filetyping when we pull archived files
>from the disk. The last threads in the flow e-mail archives relating to
>DVD are dated 1997. Has anyone used this storage technology since then?
>Thanks.

We have Retrospect (which I like), but you don't need to use Retrospect to
preserve the file attributes. Just enable Services for Macintosh on NT
Server. I have a CD burner on a Mac and I copy the flow data directly from
the NT Server shared "Mac" disk (using Chooser) onto a local hard disk (for
burning speed), then burn them using the Mac Folders and Files format in
Adaptec Toast.

DAT tapes don't have the longevity of either CD or DVD media - only a few
months for certain, and it's a real pain (and time-consuming) to hunt down
individual files for restoration. The advantage of CD-R is that if need
be, any Mac can read them. Plus, since it is write-once media, it makes it
more acceptable as an official "archive of record." DVD is indeed a
wonderful thing, and I myself lust for a DVD-writer, but until they've
adopted a reasonably stable format, I'm holding off switching to that
system for archives. I think in a year or so, they'll have hammered
something out.

--Janet


----------------------------------------
Janet E. Lewis
Computer Support (PC & Mac)
UWCCC Flow Cytometry Facility
UWCCC Imaging Facility
Clinical Sciences Center, K4/535
600 Highland Ave.
Madison, WI 53792-5666

(608)263-0313
(608)263-9226
jelewis1 at facstaff.wisc.edu
----------------------------------------



From smonard at trudeauinstitute.org Mon Apr 10 15:23:22 2000
From: smonard at trudeauinstitute.org (Simon Monard)
Date: Mon, 10 Apr 2000 16:23:22 -0400
Subject: FileGuard on MAC OS 8.6
Message-ID: <s8f1fffd.016@trudeauinstitute.org>


I have Fileguard 3.2.3 on my G3 with OS8.6, works fine

Simon

Simon Monard
FACS Lab Manager
Trudeau Institute
Saranac Lake
NY12983

Ph 518 891 3080 X352



From rburger at foothill.net Mon Apr 10 14:26:13 2000
From: rburger at foothill.net (Roger Burger)
Date: Mon, 10 Apr 2000 12:26:13 -0700
Subject: Mouse Erythroid Markers
In-Reply-To: <3.0.6.32.20000410133228.008f5ec0@pop3.unsw.edu.au>
References: <3.0.6.32.20000410133228.008f5ec0@pop3.unsw.edu.au>
Message-ID: <v04210100b517d97ce8b0@[209.77.127.42]>


Leonie Gaudry

>Hi All,
>We have been using CD 71 PE to sort for erythroid precursors but are now
>not sure that this is specific enough. What other erythroid markers are
>available for use with mice and how specific are they?
>Thank you,
>Regards Leonie


Leonie

Ter-119 reacts with mature erythrocytes and erythroid precursors and
does not react with other hematopoietic stem, lymphoid or myeloid
lineage cells . I do not know at what point in erythroid
development it is expressed but it may occur early enough for your
purpose. It is available as a PE conjugate. If you need a source
let me know.



Roger A. Burger, PhD
Technical Marketing Scientist
Miltenyi Biotec, Inc.
Ph: 530-888-8871
fax: 530-888-8925
1-800-367-6227



From bigos at stanford.edu Mon Apr 10 16:26:24 2000
From: bigos at stanford.edu (Marty Bigos)
Date: Mon, 10 Apr 2000 14:26:24 -0700
Subject: tandem conjugates
Message-ID: <v04210108b517f72af281@[171.65.21.136]>


Can someone tell me who (and when) developed the first energy
transfer tandem conjugate fluorochrome? References would be helpful
as well. Thanks.

Marty Bigos
Operations Manager
Stanford Shared FACS Facility
bigos at stanford.edu
(voice) 650-723-6959
(fax) 650-725-8564


From DOLSON4 at partners.org Mon Apr 10 13:44:26 2000
From: DOLSON4 at partners.org (Olson, Douglas)
Date: Mon, 10 Apr 2000 14:44:26 -0400
Subject: rsCD4
Message-ID: <8439A208B822D311B4DD0008C7EAAA0B019BE6C8@phsexch12.partners.org>


Does anyone know where to get recombinant soluble CD4 (to be used for in
vitro competitive binding assays)? Biogen used to make it but it seems they
don't anymore.

Thanx,
do

-----------------------------------
Douglas Olson
Experimental Hematology/AIDS Research Center
Massachusetts General Hospital
Harvard Medical School
149 13th Street
Boston, MA 02129
(617)724-2668 - Phone
(617)726-4691 - Fax
Dolson4 at partners.org



From jkalnits at vt.edu Mon Apr 10 09:38:03 2000
From: jkalnits at vt.edu (joan Kalnitsky)
Date: Mon, 10 Apr 2000 10:38:03 -0400
Subject: No subject
Message-ID: <3.0.1.32.20000410103803.00775dcc@mail.vt.edu>


Can someone provide me with Verity Software's toll free number. I have a
client interested in getting in touch with them.
Thanks in advance,
Joan K


Flow Cytometry Lab Supervisor
VMRCVM
(540) 231-4115
FAX 540-231-7367
jkalnits at vt.edu

"It is better to serve than to receive."
B. Borg


From dcdsflow at mint.net Mon Apr 10 10:03:09 2000
From: dcdsflow at mint.net (Andrea Illingworth)
Date: Mon, 10 Apr 2000 11:03:09 -0400
Subject: CD4/CD8 coexpression in pb
Message-ID: <002e01bfa2fd$e2c8cfa0$3401a8c0@andreasep>

We have noticed occasional cases with a fair amount of CD4/CD8 coexpression in peripheral bloods (up to 10% coexpression within the lymphoid gate). Most of these cases show no CMC or morphological abnormalities and we are not sure about the clinical significance of this findings. Does anybody know a "non-malignant" explanation for this finding? QC of these antibodies looks fine.

Thanks for your help!

Andrea J. Illingworth, M.S. (ASCP)
Dahl-Chase Diagnostic Services
Flow Cytometry
333 State Street
Bangor, Maine 04401

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From Sven.Nylander at astrazeneca.com Tue Apr 11 06:38:29 2000
From: Sven.Nylander at astrazeneca.com (Sven.Nylander@astrazeneca.com)
Date: Tue, 11 Apr 2000 13:38:29 +0200
Subject: pig/dog anti-CD62P
Message-ID: <3DB0284E2B7AD21189A400805F19DB2801D82937@se-hsl-mail010.hassle.se.astra.com>


Dear group
I have been trying without success to find antibodies to pig and dog CD62P
(or any other platelet activation marker)
Can anybody help me ??
Best regards
----------------------------------------------
Sven Nylander
Cell Biology and Biochemistry
AstraZeneca R&D M?lndal
431 83 M?lndal
Sweden

Phone +46 31 7762149
Fax +46 31 7763736
E-mail sven.nylander at astrazeneca.com


From rose_hughes at hgsi.com Mon Apr 10 14:53:19 2000
From: rose_hughes at hgsi.com (rose_hughes@hgsi.com)
Date: Mon, 10 Apr 2000 15:53:19 -0400
Subject: anti-hamster ab's (clarification)
Message-ID: <OFE88D4A7F.0160396F-ON852568BD.006C6292@hgsi.com>


Dear List,

Thanks for the swift responses, however, I must clarify. I am looking for
monoclonal ab's to hamster antigens, particularly MHC class I and II.....
for hamsters. If anyone knows of such a beast..........

Thanks in advance, sorry for the confusion.

Rose Hughes, B.S. CT(ASCP),(IAC),CLsp(CG)
Flow Cytometry Lab
Human Genome Sciences, Inc.
9410 Key West Ave.
Rockville, MD 20850
(301) 309-8504 ext. 2074




From davidc at ccmi.salk.edu Mon Apr 10 15:36:57 2000
From: davidc at ccmi.salk.edu (David Chambers)
Date: Mon, 10 Apr 2000 13:36:57 -0700
Subject: Cytometry Companies and the Internet
In-Reply-To: <B514E964.56B%kbahjat@ufl.edu>
References: <B514E964.56B%kbahjat@ufl.edu>
Message-ID: <00041013453601.11873@xena.salk.edu>


Hear, Hear!

Keith has just voiced the collective opinion.

What about it, Cytometer Companies? Why so hesitant to move into the
21st Century? Most software companies put their product on the web these days,
why not you too? Saves a lot of $$$$ (hint, hint!) and aggro.


On Sat, 08 Apr 2000, Keith Bahjat wrote:
> (snippety snip)
> As the software that controls our cytometers, like Cellquest and EXPO, require
> hardware dongles, and thus are VERY secure, why do the companies still make
> us jump through hoops to get updated copies?? They could post the update on
> a web site, and save the cost of having those 5000 CD's burnt every time you
> upgrade to version X.X.1.
> (snippety snip)


From wenre at pei.de Mon Apr 10 04:20:11 2000
From: wenre at pei.de (Renate Wenig)
Date: Mon, 10 Apr 2000 11:20:11 +0200
Subject: Vital-staining
Message-ID: <s8f1b90d.037@SV-BK-02.PEI.DE>


My name is Renate Wenig from the Paul-Ehrlich-Institut in Langen/Germany.
I'm FACS beginner.
Is there anybody with experience in vital-staining of adherent cells for
FACS-measerments?
I want to count absolute cell-number of vital cells with FACS.
Until now I try to stain the dead cells with PI but it doesn't work:
After detaching cells from the flaks bottom with EDTA all cells are PI positiv in FACS.
Can anybody give me useful tips or instructions for vital-staining of adherent cells?
Or can anybody say me where I can read about itMy E-mail adress is: wenre at pei.de
Thank you for answering!



From jbrewer at hsc.wvu.edu Mon Apr 10 14:29:22 2000
From: jbrewer at hsc.wvu.edu (Jamie Brewer)
Date: Mon, 10 Apr 2000 15:29:22 -0400
Subject: annexin V stain questions
Message-ID: <s8f1f36d.072@gateway.hsc.wvu.edu>


I am writing to inquire about an annexin V stain I am used today from Pharmingen. After
calling the techinical support service as Pharmingen, I was given this E-mail
address to correspond to with my unanswered question. The confusion comes in that
the Pharmingen protocol does not have me wash off any unbound annexin prior to flow
cytometry analysis. This is completely contradictory to any fluorescent stain I have ever
used. Typically, the unbound stain fluoresces when analyzed, losing any specificity the
system had. My results seemed to turn out as expected today but I don't understand why
the unbound stain is not causing me a problem. I have been unable to find any reference
to the annexin being "active" only in the bound form as opposed to the unbound form
or any statement indicating the something in the binding buffer inactivates unbound
annexin. The technician at Pharmingen didn't have any idea why the unbound stain is
not causing a problem. Do you have any understanding of this? Also, the protocol does
not offer any option to fix the cells, something which I really need to do since I
am using potentially infectious human cells in a common flow cytometry facility. The
Pharmingen representative said that she knew of some people who had fixed the cells
and it worked alright but not as well as if the cells were not fixed. Do you have
any advice/suggestions?

Please correspond to:
Jamie Brewer
jbrewer at hsc.wvu.edu
Microbiology/Immunology Department, Mary Babb Randolph Cancer Center, West Virginia
University


From bunny at cotleur.com Mon Apr 10 16:51:01 2000
From: bunny at cotleur.com (bunny)
Date: Mon, 10 Apr 2000 17:51:01 -0400
Subject: analysis of few cells
Message-ID: <38F24CC3.A3D636E2@cotleur.com>


Fellow Flowers:
I wonder if I could gather some opinions on releveance as it relates to
minimum numbers of events collected.
I am assisting a lab in developing FACS analysis of PBMC's and monocytes
in CSF. They informed me that when they run FACS, they are "lucky" to
get 100 events. I raised the questions of relevance, stating that those
100 "events" could as easily contain some non-cell events as cell
events. They insist their data is good becasue it compares somewhat (?)
with a lab in Europe they are collaborating with. We have had many
conversations on this.
(I should mention- neither lab has direct FACS experience. They got all
their protocols second hand, and just collect what they collect).
Additional information: they are looking at chemokine expression
(another difficult task) on these few cells- and I believe they are
using the PBMC's to set up isotype controls. I doubt they even use comp controls.

Please throw your 2cents my way. And if any of you do this type of
analysis (few cells) with alternative techniques- I'd love to hear about it!
Thanks-
--



Bunny






*******************************************************
Bunny Cotleur +*+ Bunny Cotleur
Cleveland Clinic Foundation *+* 2001 Lester RD
Neurosciences NC30 +*+ Valley City, OH 44280
9500 Euclid Avenue *+* 330-483-4800
Cleveland, OH 44195 +*+ bunny at cotleur.com
216-444-1164 *+*
cotleua at ccf.org +*+

*******************************************************
When you do something, you should burn yourself completely, like a good
bonfire, leaving no trace of yourself.
(Shunryu Suzuki)


From djyoung at UCSD.Edu Mon Apr 10 13:48:21 2000
From: djyoung at UCSD.Edu (Dennis J. Young)
Date: Mon, 10 Apr 2000 11:48:21 -0700
Subject: sample/sheath ratio after sort
Message-ID: <3.0.1.32.20000410114821.00824e30@popmail.ucsd.edu>


The general estimate would be how much sample is used per hour divided by
how much sheath is used.
Or assuming 1 microliter per second of sample is used and that each drop
has 3.53 times the cube of the diameter of the nozzle, so 10^6 drops equals
3.53 ml for a 100 micron nozzle. (NOT 0.523 ml)
If the pressure is only around 12 psi, then the frequency of drops could be
say roughly 2.8 10^4 drops per second)
For 35 seconds (10^6 over 2.8 X 10^4):
35 microliters sample per 3.53 ml sheath.
So the sample is around 1% of the sorted droplet.

"A jet vibrated at the optimum frequency for droplet generation
(lambda=4.5d) will produce droplets of volume 3.53d^3. If they were
spherical, their diameter would be 1.9 times that of the jet."
page 103, Flow Cytometry: Instrumentation and Data Analysis. Eds. Van
Dilla, MA, Dean PN, Laerum OD, Melamed MR. Academic Press, 1985.

>At 05:40 PM 4/5/00 -0500, you wrote:
>>
>>Hi cytometry fans,
>>
>> I have a client that wants to know how much of the drop volume will be
>>taken up by the sample fluid (I need a general estimate). We will be using
>>an ELITE sorter (12 PSI) with 100 micron sort sense tip to sort. To make my
>>life easier, has anyone calculated the sample/sheath ratio after sorting at
>>a specific sample input pressure? I thought about running a concentrated
>>blue dye and sorting under conditions similar to the eventual client sort
>>and then measuring the OD in a spec original sample vs. sorted sample. Has
>>anyone tried this and was it possible to see any absorbance in the diluted
>>sort sample? Looking forward to your replies,
>>
>>Kristi Harkins

---
Dennis

Dennis J. Young
Mail:<<mailto:djyoung at ucsd.edu>>
WWW:<<http://cancer.ucsd.edu/SResources/flow.htm>>

Flow Cytometry Core Facility
University of California, San Diego
Internal Medicine Group, Bldg #4, Rm 126
9500 Gilman Drive La Jolla CA 92093-0671
Telephone:(858) 822-0407


From RFischer at therimmune.com Tue Apr 11 08:09:50 2000
From: RFischer at therimmune.com (Fischer, Randy)
Date: Tue, 11 Apr 2000 09:09:50 -0400
Subject: Cytometry Companies and the Internet
Message-ID: <A01B29D8827CD311ADF10008C79F3DBF02B51F@labsrv.therimmune.com>


Good point Keith. It would then eliminate what happened at one of my
previous positions where BD never sent us the upgrades until we had a
problem with the software. Then, they were surprised at the version I
had. If there was an update link on their website, that would not
happen.

Randy Fischer
TherImmune Research Corporation
9700 Great Seneca Hwy
Rockville, MD 20850
(240) 453-6256
RFischer at therimmune.com

> ----------
> From: Keith Bahjat
> Sent: Saturday, April 8, 2000 10:49 AM
> To: Cytometry Mailing List
> Subject: Cytometry Companies and the Internet
>
> Which raises an interesting point we were discussing earlier this
> week. As the software that controls our cytometers, like Cellquest and
> EXPO, require hardware dongles, and thus are VERY secure, why do the
> companies still make us jump through hoops to get updated copies??
> They could post the update on a web site, and save the cost of having
> those 5000 CD's burnt every time you upgrade to version X.X.1. The
> WHOLE CD with all of BD's sample files and documentation on it is
> still only 40 Mb! That's not even a 7 minute download at only
> 100K/sec.
>
> Hope someone out there is listening. I think we've all got enough
> useless AOL, Compuserve, and Earthlink CD's laying around. We don't
> need CD's from cytometry companies on top of that. Post it on a web
> site.......PLEASE!
>
> kb
>
> Keith Bahjat
> kbahjat at ufl.edu
>
>
>
> on 4/7/00 4:28 PM, Joanne Thomas at thomas at tritechinc.com wrote:
>
>
>
> Hi All!
> We have been trying to purchase the new version of CellQuest
> which is supposed to address the OS 9 issues and have been told that
> it is on back order for some time now. Has anyone else had a problem
> getting version 3.3???
>
> Joanne Thomas, M.S.
> Director of Operations
> TRITECH Field Engineering
> 2014 Renard Court, Suite I
> Annapolis, MD ?21401
> 1-800-886-7004 (USA)
> 1-410-266-1522
> 410-266-0935 (FAX)
> thomas at tritechinc.com
>
>
>
>
>


From Darley at cardiff.ac.uk Tue Apr 11 12:29:51 2000
From: Darley at cardiff.ac.uk (Richard L. Darley)
Date: Tue, 11 Apr 2000 12:29:51 GMT0BST
Subject: 2 post-doctoral positions in the UK
Message-ID: <5CFC9454B76@NPRDCF1R.CF.AC.UK>


I would like to draw attention to the following research positions in
this Department. One of these positions in particular would be very
suitable for those with experience of flow cytometric analysis as it
will involve multiparameter analysis of retrovirally-transduced
primary haematopoietic cells. If you are interested please feel free
to contact me directly for any informal inquiries.<bold>


UNIVERSITY OF WALES COLLEGE OF MEDICINE


2 POST DOCTORAL RESEARCH FELLOWS


Starting Salary £16,286-£20,811 per annum


Two 3-year post doctoral research positions have become
available within the Department of Haematology, one funded
by the Leukaemia Research Fund and one funded by a
UWCM endowment fund. These positions will form part of a
significant new initiative within the Department to
investigate the effects of the t(8:21) translocation on
haematopoietic development. The study, which will be
carried out in collaboration with Dr James Downing (St
Jude's Hospital, Memphis) has 2 principle aims: first, to
investigate the effects of this translocation on primary cell
development using a model system already established (<italic>J.
Exp. Med. 185, 1337 1997)</italic> and second, to analyse changes in
gene expression arising as a consequence of this
translocation. In parallel with these studies we will also
examine the efficacy of potential therapeutic agents in
targeting t(8:21) disease. Experience in molecular and/or
cell biology will be advantageous as will the ability to
supervise the work of both students and research assistants.


An application pack (quoting Reference Number MCH418 is
available from the Personnel Department, University of
Wales College of Medicine, Heath Park, Cardiff CF14 4XN;
telephone 029 2074 5110 (24 hour answer phone); email
uwcm_pers at cf.ac.uk.



<nofill>
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
PLEASE NOTE CHANGES TO CONTACT NUMBERS AND POST CODE
From: Dr Richard Darley, Ph.D.
Department of Haematology (A7)
University of Wales College of Medicine
Heath Park
Cardiff, CF14 4XN
U.K.
tel +44 (0)29 20 74 34 85
or +44 (0)29 20 74 23 75
Fax +44 (0)29 20 74 46 55
e-mail: darley at cf.ac.uk


From aa9080 at wayne.edu Mon Apr 10 10:38:26 2000
From: aa9080 at wayne.edu (Eric Van Buren)
Date: Mon, 10 Apr 2000 11:38:26 -0400
Subject: DVD-RAM for data storage
In-Reply-To: <3.0.32.20000406155212.006e9590@flocyt.int-med.uiowa.edu>
Message-ID: <l03130300b5178f5d8c1c@[146.9.12.118]>


Justin Fishbaugh,

About a year ago, we did exactly what you outlined in your message. Now
every evening, Retrospect runs on a G3/400 and backs up data from the NT
server as well as the acquisition and analysis Macs and PCs. The data is
short-term archived to DVD-RAM. For long-term archival, the data from
DVD-RAM is written to CD-r (two copies). Some trials and tribulations
follow.

The NT server has just one 10GB disk, partitioned into 4 smaller disks (NT
4.0 can't handle such a big disk in one piece). Installing NT 4.0 Service
Pack 4 (SP4) was not easy; first you install NT 4.0, then apply the
"Service Pack" updates and "optional" software (like the internet software,
including the ftp server) in the specified order, then read all sorts of
technical notes from the maze-like Microsoft website when things go wrong.
After your 3rd install, you should have a running system. (Hopefully
Windows NT 2000 isn't as much of a "beta" software as NT4SP4 was, but I
wouldn't hold my breath.)

Since NT uses AppleTalk to communicate with Mac clients, and our university
does not support AppleTalk routing, we knew we would have trouble with our
remote sites. I chose DAVE software to get around this roadblock, but it
was expensive. Fetch and ftp will also work, but you will lose the
CellQuest signature.

We also purchased Netscape Calendar Server (NCS) for NT for our online
scheduling. This system just became active, and we haven't had much trouble
using the software. The bigger problem was installing and setting it up. It
turned out that the version of NCS we originally purchased was not
compatible with NT4SP4; it wanted SP3, and did not "play nicely" with IIS
(NT's internet services). Sun/Netscape finally released a newer version for
SP4.

I chose NT over unix because I thought NT would be easier to set up. In
hindsight, I realize that this is probably not the case. Cost and
compatibility were other factors. However, Linux still looks tempting ...
and even Mac OS X may be a contender now.

Retrospect works well, but we're still having difficulties reaching a
remote site because of a firewall. I talked to Dantz and the local IT
responsible for the firewall, but evidently this is not as easy to fix as I
was lead to believe. In order to keep the CellQuest signature, you will
have to mount the NT server as an AppleShare drive on the Mac, so make sure
AppleTalk can make the trip between your Mac and NT server.

We use a combination of DVD-RAM and CD-r because we already had the CD-r
drive. I wanted a backup drive for Retrospect that used large-capacity
re-writable media, but I personally don't like tape, so I got the DVD-RAM
drive as part of a new G3.

It should be noted, however, that DVD-RAM has a capacity of 5.2GB per disk,
but only 2.6GB per each SIDE of the disk. Unless your DVD-RAM drive has two
read/write heads (if such a drive even exists) you will only be able to
access 2.6GB at any one time. (You unmount the disk, and physically flip it
over to access the other 2.6GB.)

Eric


>Greetings,
>
>We are in the process of switching to a larger and faster data server
>(Windows NT) to store our flow cytometry data. We will be using either one
>or two 9GB drives. I am wrestling with long term archive options that
>currently include 4mm DAT tape, CD-RW/CD-R or DVD-RAM. I am leaning toward
>DVD-RAM because of the 5.2GB capacity and longevity of the media. The down
>side is that DVD-RAM is not a mature technology like CD-RW/CD-R or 4mm DAT.
> We'll probably install it on a fast MAC in the lab (the server is in
>another building) and use Retrospect backup software so that we can retain
>Cellquest file attributes without Filetyping when we pull archived files
>from the disk. The last threads in the flow e-mail archives relating to
>DVD are dated 1997. Has anyone used this storage technology since then?
>Thanks.
>
>
>
>
>------------------------------------------------
>| Justin Fishbaugh |
>| University of Iowa |
>| Flow Cytometry Facility |
>| 48 EMRB |
>| Iowa City, IA 52242 |
>| |
>| justin-fishbaugh at uiowa.edu |
>| http://www.medicine.uiowa.edu/flowcytometry/ |
>------------------------------------------------


/\/\/\_ Eric Van Buren, aa9080 at wayne.edu
\ \ \ Karmanos Cancer Institute and Immunology & Microbiology
\_^_/ Wayne State University, Detroit, Michigan, USA




From rburger at foothill.net Mon Apr 10 14:50:22 2000
From: rburger at foothill.net (Roger Burger)
Date: Mon, 10 Apr 2000 12:50:22 -0700
Subject: job announcement
Message-ID: <v04210102b517e0eba630@[209.77.127.42]>

Miltenyi Biotec, Inc
251 Auburn Ravine Road, Ste 208
Auburn, CA 95603
Phone: 1-530-888-8871 or 1-800-367-6227
Fax: 1-530-888-8992



Technical Marketing Scientist

Miltenyi Biotec, a growing corporation in the field of Magnetic
Separation, is seeking a Technical Marketing Scientist. In this
position you will consult with customers and technical
representatives on technical issues involving technique, protocols
and general product applications in Magnetic Separation.
Responsibilities include developing and teaching new applications and
techniques, writing protocols, newsletters and making professional
presentations.

The ideal candidate will have an advanced degree in Immunology,
Hematology or Cell Biology with a broad background in the Life
Sciences. Lab experience and strong communication capabilities are a
must. Candidates with an undergraduate degree and strong lab
experience are also encouraged to apply.

If you wish to discuss this exciting opportunity, call Mindy
Wilke-Douglas at Miltenyi Biotec. You may also fax or e-mail your CV
for immediate attention (mindy at miltenyibiotec.com). Miltenyi Biotec
is an equal opportunity employer.

Roger A. Burger, PhD
Technical Marketing Scientist
Miltenyi Biotec, Inc.
Ph: 530-888-8871
fax: 530-888-8925
1-800-367-6227
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From rhester at jaguar1.usouthal.edu Tue Apr 11 13:48:44 2000
From: rhester at jaguar1.usouthal.edu (ray hester)
Date: Tue, 11 Apr 2000 13:48:44 -0500
Subject: Activated-Macrophage Markers
Message-ID: <LPBBKDFDLBFPLGEIAJEGKEHJCAAA.rhester@jaguar1.usouthal.edu>



Hi,

Are there markers that can be used to distinguish activated alveolar
macrophages in BAL and that are detectable by FACS, i.e., not soluble
factors from culture supernates?

Thanks for any help.

Ray Hester
Univ. South Alabama



From Howard.Robinson at joslin.harvard.edu Tue Apr 11 11:29:14 2000
From: Howard.Robinson at joslin.harvard.edu (Robinson, Howard)
Date: Tue, 11 Apr 2000 12:29:14 -0400
Subject: Precautions for Sorting Live Human Samples
Message-ID: <047232CBD5F8D21194240008C7F3625804D681D0@josmail.harvard.edu>


I know some researchers that are interested in sorting live samples of human
peripheral blood and possibly other types of samples. I'm interested in
finding out what I should be concerned about as far as aerosol or other
means of transmission of disease to myself and others in the room during
sorting. What precautions should I take/require? What screening should I
require prior to accepting samples for sorting? I was hoping that someone
could point me in the right direction.

Any help is appreciated.

Howard Robinson


From hms at shapirolab.com Tue Apr 11 15:32:50 2000
From: hms at shapirolab.com (Howard Shapiro)
Date: Tue, 11 Apr 2000 16:32:50 -0400
Subject: tandem conjugates
In-Reply-To: <v04210108b517f72af281@[171.65.21.136]>
Message-ID: <4.2.0.58.20000411163039.009e17b0@shell1.shore.net>


Marty Bigos asks-


>Can someone tell me who (and when) developed the first energy
>transfer tandem conjugate fluorochrome? References would be helpful
>as well. Thanks.

Alex Glazer and Lubert Stryer, in the early 1980's; it was PE-APC, which
has recently been resurrected.

Glazer AN, Stryer L: Fluorescent tandem phycobiliprotein
conjugates. Emission wavelength shifting by energy transfer. Biophys J
43:383, 1983

-Howard





From gap at MIT.EDU Tue Apr 11 13:31:23 2000
From: gap at MIT.EDU (Glenn Paradis)
Date: Tue, 11 Apr 2000 14:31:23 -0400
Subject: non fluorescent beads
Message-ID: <v03020900b5191efdd872@[18.114.0.62]>




Hi Everyone,

Does anyone have a cheap source of non fluorescent 2-10um beads? I want to
run some hi pressure test sorts at rates of 30,000-50,000 beads/sec and do
not want to pay a fortune for them.


Thanks,

Glenn
MIT




From helix.mgh.harvard.edu at helix.mgh.harvard.edu Tue Apr 11 16:20:44 2000
From: helix.mgh.harvard.edu at helix.mgh.harvard.edu (David Dombkowski)
Date: Tue, 11 Apr 2000 16:20:44 -0500
Subject: Antibody to mouse P21 or P27
Message-ID: <v03007801b51947048924@[132.183.148.45]>


I am looking for an antibody either fluorochrome conjugated or not
specific for mouse P21 or P27. Please let me know if this is available.

David M. Dombkowski
dombkowski at helix.mgh.harvard.edu
Flow Cytometry-Pathology-CNY rm7017
149 13th Street
Massachusetts General Hospital-East
Boston, MA 02129
Tel. (617)726-1683
Fax.(617)724-3164





From vvestereng at mail.cc.nih.gov Tue Apr 11 14:00:54 2000
From: vvestereng at mail.cc.nih.gov (vvestereng@mail.cc.nih.gov)
Date: Tue, 11 Apr 2000 15:00:54 -0400
Subject: DNAse
Message-ID: <v04011704b518f0d3028f@[128.231.81.42]>


Hi everybody!

I was wondering if anyone out there could help me with a problem?

I would like to use DNAse I to brake up clumps of lymphocytes from mouse
spleens. I understand that most people have been using DNAse I with a
concentration of 20.000-100.000 U/ul. I have tried to find a company
selling it, but most companies earlier selling these products seem to have
discontinued them. Does anyone know a company still selling it.



Thanks

Vibeke H. Vestereng, MD
Critical Care Medicine Dept.
National Institutes of Health
10 Center Drive, Rm 7D43
Bethesda
MD 20892-1662
Phone: 301-435-2315
FAX: 301-480-3389
E-mail: vvestereng at mail.cc.nih.gov


From shawn_jackson at student.hms.harvard.edu Wed Apr 12 09:19:20 2000
From: shawn_jackson at student.hms.harvard.edu (shawn_jackson)
Date: Wed, 12 Apr 2000 10:19:20 -0400
Subject: annexin V dim cells
Message-ID: <38F3C4E9@webmail.student.med.harvard.edu>


hey flow-ers

I have a question concerning the occurrence of annexin V-dim cells vs. bright
cells. I need help with references and/or data experience!

I am staining peripheral blood from mice infected with vaccinia, and then
staining the CD8 cells with annexin V-fitc. I see a population of annexin+
cells extending even up past the 2nd log at many time points, but at certain
times I see very bright cells up at the 3rd log.

I have been warned that only these very bright cells are truly AV+, cells with
any intentions whatsoever of doing anything related to a death process, while
the dimmer cells represent actively proliferating cells only, with no
intentions of dying. and these dimmer cells are likely to return to an AV-
population quickly.

Could anyone help me with references or personal experince addressing the
status of these AV dim cells in contrast to bright ones?

thanks!

shawn jackson
grad student
harvard univ
boston, ma



From dpr4w at virginia.edu Tue Apr 11 09:33:48 2000
From: dpr4w at virginia.edu (D Raymond)
Date: Tue, 11 Apr 2000 16:33:48 +0200
Subject: Quantification of macrophage phagocytosis
Message-ID: <01BFA3D3.BBB39A40@bootp-62-199.bootp.Virginia.EDU>


I am trying to quantify macrophage bacterial phagocytosis but have had numerous problems.
I am trying to quantify the phagocytosis of E. coli by the murine macrophage cell line
RAW 264.7. I have tried using FITC labelled E. coli but have had problems quenching
external bacteria. The RAW cells take up the quenching agent (ethidium bromide) almost
immediately They do appear viable when I quantify them by tryphan blue exclusion and,
under similar conditions, we see significant cytokine production. If anyone has any
experience with this technique I would appreciate any input. Thank you for your time.
-Daniel Raymond (Charlottesville, VA)



From Sergey.Krylov at ualberta.ca Wed Apr 12 09:07:09 2000
From: Sergey.Krylov at ualberta.ca (Sergey)
Date: Wed, 12 Apr 2000 08:07:09 -0600
Subject: Analysis of few cells - Chemical Cytometry is an alternative
References: <38F24CC3.A3D636E2@cotleur.com>
Message-ID: <38F4830D.F458A42C@UAlberta.Ca>


Dear Colleagues,

I think that Chemical Cytometry can be an alternative to flow cytometry for those in
the cytometry community who have to deal with very few cells (<1000). Chemical
Cytometry is our term to describe the multi-component chemical analysis of single
cells using the separation and detection tools of instrumental analytical chemistry.
We are using Capillary Electrophoresis (CE) with Laser-Induced Fluorescence (LIF)
detection for this purpose. In essence in chemical cytometry, a single cell is
injected into a separation capillary, lysed inside the capillary and the cellular
contents are then separated using CE and monitored at the opposite end of the
capillary using an LIF detector. We described the concept of Chemical Cytometry and a
prototype instrument in Anal. Chem. 2000, 72, 872-877 (ask me to send a pdf file if
interested). Introducing the separation step allows for a multi-component analysis of
the cellular contents (theoretically hundreds of components). In addition the
technique is characterized by a very decent sensitivity - LIF routinly provides the
detection limits of ~100 molecules. Thus, the amount of information one can obtain on
a single cell using Chemical Cytometry is considerably superior to that in flow
cytometry. Another advantage of Chemical Cytometry is its easy coupling with
fluorescence image cytometry to monitor cell's morphology or physiological status
prior to the Chemical Cytometry analysis. I have recently used this approach to
correlate the cell cycle with a number of enzymatic activities. Such a combination
allows to recognize the subpopulations of cells with unique metabolic patterns within
the same phase of the cell cycle (see Cytometry 1999, 37, 14-20). Of course, CE is
associated with time consumption incomparable with that in flow cytometry (although
the amount of information gathered may compensate completely this limitation). I am
currently proposing to develop a multi-capillary Chemical Cytometry instrument (think
of the PE Applied Biosystems 96-capillary DNA sequencers being used by Celera for
human genome project) to considerably increase the throughput.

If anybody interested in using such an approach I would be happy to help. I would
also appreciate any kind of feedback from the cytometry community.

Sergey


bunny wrote:

> Fellow Flowers:
> I wonder if I could gather some opinions on releveance as it relates to
> minimum numbers of events collected.
> I am assisting a lab in developing FACS analysis of PBMC's and monocytes
> in CSF. They informed me that when they run FACS, they are "lucky" to
> get 100 events. I raised the questions of relevance, stating that those
> 100 "events" could as easily contain some non-cell events as cell
> events. They insist their data is good becasue it compares somewhat (?)
> with a lab in Europe they are collaborating with. We have had many
> conversations on this.
> (I should mention- neither lab has direct FACS experience. They got all
> their protocols second hand, and just collect what they collect).
> Additional information: they are looking at chemokine expression
> (another difficult task) on these few cells- and I believe they are
> using the PBMC's to set up isotype controls. I doubt they even use comp controls.
>
> Please throw your 2cents my way. And if any of you do this type of
> analysis (few cells) with alternative techniques- I'd love to hear about it!
> Thanks-
> --
>
> Bunny
>
> *******************************************************
> Bunny Cotleur +*+ Bunny Cotleur
> Cleveland Clinic Foundation *+* 2001 Lester RD
> Neurosciences NC30 +*+ Valley City, OH 44280
> 9500 Euclid Avenue *+* 330-483-4800
> Cleveland, OH 44195 +*+ bunny at cotleur.com
> 216-444-1164 *+*
> cotleua at ccf.org +*+
>
> *******************************************************
> When you do something, you should burn yourself completely, like a good
> bonfire, leaving no trace of yourself.
> (Shunryu Suzuki)



--
Sergey Krylov, Ph.D.
Department of Chemistry
University of Alberta
Edmonton, Alberta T6G 2G2
Canada

Phone: 780-492-9251
Fax: 780-492-8231
E-mail: Sergey.Krylov at UAlberta.Ca
Web: http://www.chem.yorku.ca/profs/krylov/




From simmmmer at yahoo.com Tue Apr 11 13:01:06 2000
From: simmmmer at yahoo.com (Maciej Simm)
Date: Tue, 11 Apr 2000 11:01:06 -0700 (PDT)
Subject: Human B cell markers
Message-ID: <20000411180107.1529.qmail@web2005.mail.yahoo.com>


Greetings everyone,

What (if any) antigens are expressed on B cells in similar amount to
Leu-12 (CD19)? Are the two forms of CD80 (B7 and Bsomethingelse)
expressed equally?

Also what is a good reference for the change of expression of various
molecules on B cells?

Finally, more trivial, what is the "leu" nomenclature? is "cd"
nomenclature more common now?

Thanks!

PS Anyone who answers gets an electronic cookie.

=====
`---------------------------------------------`
| Maciej S. Simm | 525 E 68th Street |
| Research Technician | Room N-805 |
| Cornell Medical Center | Tel. 212.746.3428 |
`---------------------------------------------`
| www.cd4cd8.com |
`---------------------------------------------`

__________________________________________________
Do You Yahoo!?
Talk to your friends online with Yahoo! Messenger.
http://im.yahoo.com


From jpr at flowcyt.cyto.purdue.edu Tue Apr 11 15:19:41 2000
From: jpr at flowcyt.cyto.purdue.edu (jpr@flowcyt.cyto.purdue.edu)
Date: Tue, 11 Apr 2000 15:19:41 -0500
Subject: Labtek Dishes problem
Message-ID: <38F3428D.15846.169CB54@localhost>


Colleagues
We have been using Labtek (made by Nunc) Chambered
Coverglass dishes (8 chambers) for several years (#136439). [see a
picture on
http://www.cyto.purdue.edu/flowcyt/confocal/cfcl7.htm

We found out recently that they have redesigned all their plates, by
making small (but significant physical size changes) They also
changed the glass bases - bottom line is the new dishes don't fit
any of our specially designed heatsinks, holders, etc. They also
stuck a "handle" on top of the clear plastic lis, so now you cant
image through the dishes. Real smart people must have just taken
over at Labtek!!!!

So, if anyone has some of the old dishes (sterile packs of course) in
stock, we love to hear from you, we will be happy to exchange equal
value, give up a graduate student or two, or a few bottles of nice
plonk.....whatever it takes.

If anyone out there has any influence with Nunc, I think they pretty
much destroyed a really successful product....!!!

HELP please....
thanks
Paul Robinson

J.Paul Robinson, PhD PH:(765)4940757
Professor of Immunopharmacology
Professor of Biomedical Engineering
Purdue University FAX:(765)4940517
EMAIL:jpr at flowcyt.cyto.purdue.edu
WEB: http://www.cyto.purdue.edu


From CRAIGF at uthscsa.edu Tue Apr 11 16:57:14 2000
From: CRAIGF at uthscsa.edu (Craig, Fiona E)
Date: Tue, 11 Apr 2000 16:57:14 -0500
Subject: CD4/CD8 coexpression in pb
Message-ID: <3B6B9A9E977ED311B10500A0C9B41269414B45@oryx.uthscsa.edu>


Nah EH, King D, Craig FE.
CD4 and CD8 Antigen Co-expression in Clinical Specimens.
Archives of Pathology and Laboratory Medicine 121:381-384,
1997.


> ----------
> From: Andrea Illingworth
> Sent: Monday, April 10, 2000 10:03 AM
> To: Cytometry Mailing List
> Subject: CD4/CD8 coexpression in pb
>
> We have noticed occasional cases with a fair amount of CD4/CD8
> coexpression in peripheral bloods (up to 10% coexpression within the
> lymphoid gate). Most of these cases show no CMC or morphological
> abnormalities and we are not sure about the clinical significance of this
> findings. Does anybody know a "non-malignant" explanation for this
> finding? QC of these antibodies looks fine.
>
> Thanks for your help!
>
> Andrea J. Illingworth, M.S. (ASCP)
> Dahl-Chase Diagnostic Services
> Flow Cytometry
> 333 State Street
> Bangor, Maine 04401
>
>


From guo at mm11.ukl.uni-freiburg.de Tue Apr 11 16:02:01 2000
From: guo at mm11.ukl.uni-freiburg.de (Guo Yalin)
Date: Tue, 11 Apr 2000 22:02:01 +0100
Subject: help
Message-ID: <A7E3DF51154@mm11.ukl.uni-freiburg.de>


Dear everybody,

I am doing sorting SP cells with Hoechst 33342 from human
hematopoietic cells, it's a novel way to sort human hematopoietic
stem cells. I have been sticking with Dr. Goodell's protocol and got
nice SP cells as I presumed. But I have some problem to confirm the
SP cells. As I recommended, I used Verapamil, a G-protein blocked to
stain with Hoechst. I don't know what the SP cells will be changed
if Verapamil is used, in short, I don't know how to confirm human SP
cells. Could you be so kind to help me?

Wish to hear from you!

Yours sincerely,

Yalin Guo

Hematology & Oncology Department
University Freiburg
Freiburg, Germany

email: guo at mm11.ukl.uni-freiburg.de
Yalin Guo
Nothnagel Lab
Hematology/Onkology Department
University of Freiburg
Hugstetter Strasse 55
D-79106 Freiburg
Germany
Tel: +49 761 270 7199(LAB)
Fax +49 761 270 7177
Email: guo at mm11.ukl.uni-freiburg.de


From donwalker at chiroscience.com Tue Apr 11 14:10:07 2000
From: donwalker at chiroscience.com (Walker, Don (SEA))
Date: Tue, 11 Apr 2000 12:10:07 -0700
Subject: CD4/CD8 coexpression in pb
Message-ID: <57E4F9D1FB28D311B6FB009027700F651B0CEB@xch_sea_01.darwin>


Andrea,
Are these cells PI Neg.?
?
Don
Celltech-Chiroscience?
Bothell, WA
Phone: 425.489.8110
?

-----Original Message-----
From: Andrea Illingworth [mailto:dcdsflow at mint.net]
Sent: Monday, April 10, 2000 8:03 AM
To: cyto-inbox
Subject: CD4/CD8 coexpression in pb


We have noticed occasional cases with a fair amount of CD4/CD8
coexpression in peripheral bloods (up to 10% coexpression within the
lymphoid gate).? Most of these cases show no CMC or morphological
abnormalities and we are not sure about the clinical significance of
this findings.? Does anybody know a "non-malignant" explanation for this
finding?? QC of these antibodies looks fine.
?
Thanks for your help!
?
Andrea J. Illingworth, M.S. (ASCP)
Dahl-Chase Diagnostic Services
Flow Cytometry
333 State Street
Bangor, Maine 04401




From steve at habanero.cb.uga.edu Tue Apr 11 17:06:57 2000
From: steve at habanero.cb.uga.edu (Steve G. Hilliard)
Date: Tue, 11 Apr 2000 18:06:57 -0400 (EDT)
Subject: Vital-staining
In-Reply-To: <s8f1b90d.037@SV-BK-02.PEI.DE>
Message-ID: <Pine.LNX.4.10.10004111752480.28468-100000@habanero.cb.uga.edu>


Renate,

PI is normally excluded from intact (living) cells, but I would imagine
the EDTA is damaging the cell membranes enough to allow the PI
to enter. This is also a big problem with cells that stick so well they
have to be scraped off the flasks.

I don't know that anyone has done this, but you might try EMA (ethidium
monoazide), incubating the cells with it while they are still adherent.
When irradiated by bright white light (fluorescent lamp) it irreversibly
binds to the DNA, and the unbound dye will be flushed out of the
living cells during a PBS wash step. Then you could try the EDTA removal.
The original citation for EMA is Riedy et al, 1991. Cytometry 12:133-139,
but after that I think you would be blazing new trails. I suppose
you might want to use dishes instead of culture flasks (so you
could remove the top during irradiation) but it might not matter.

I hope this helps!

Steve
+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
Steve G. Hilliard (706) 542-9474
University of Georgia Cell Analysis Facility
flowman at uga.edu http://floweb.cb.uga.edu/

On Mon, 10 Apr 2000, Renate Wenig wrote:

>
> My name is Renate Wenig from the Paul-Ehrlich-Institut in Langen/Germany.
> I'm FACS beginner.
> Is there anybody with experience in vital-staining of adherent cells for
> FACS-measerments?
> I want to count absolute cell-number of vital cells with FACS.
> Until now I try to stain the dead cells with PI but it doesn't work:
> After detaching cells from the flaks bottom with EDTA all cells are PI positiv in FACS.
> Can anybody give me useful tips or instructions for vital-staining of adherent cells?
> Or can anybody say me where I can read about itMy E-mail adress is: wenre at pei.de
> Thank you for answering!
>




From juergen.ostwald at medizin.uni-rostock.de Wed Apr 12 06:20:13 2000
From: juergen.ostwald at medizin.uni-rostock.de (Juergen Ostwald)
Date: Wed, 12 Apr 2000 13:20:13 +0200
Subject: CD4/CD8 coexpression in pb
Message-ID: <38F45BED.8137E62E@medizin.uni-rostock.de>


Dear Andrea,
we also have found some CD4+CD8+ double positive lymphocytes in a lot of
probes from human pb via flowcytometry. For my knowlegde these cells are
normal lymphocytes which are exported from thymus where in selection
processes some mistakes occurs, resulting in some 4/8 double positive
cells.
>From our experience these cells are rising up after activation of the
immunsystem, like and paralleled by CD3+HLA-DR+.
For example we have the following numbers in peripheral blood
lymphocytes of controll adults and ENT-Cancer patients in CD4+/CD8+
lymphocytes (%; U-Test):
Control : 2,38 (n=32)
p= 0.002

Patients with acute cancer : 4,19 (n=73)
p= 0.003

Patients after therapy and
cured : 2,97 (n=112)
p= 0.04
Patients with recurrence : 4,25 (n=30)

My be, this is helpfull for you.

J.Ostwald
ENT-Clinic
University Rostock
Germany




From bunny at cotleur.com Tue Apr 11 21:34:18 2000
From: bunny at cotleur.com (bunny)
Date: Tue, 11 Apr 2000 22:34:18 -0400
Subject: analyzing low cell numbers:Part2 (more details!)
Message-ID: <38F3E0A5.63F107E3@cotleur.com>


Thank you to all that responded re: low # cells to analyze. (30
responses day one! I sure can pick the topic,eh?)
I see from the varied responses I received I was waaaay too vague on
what I was asking. So please allow me to re-post this query a bit more detailed.

My friends are looking at chemokine expression in monocytes in CSF of MS
patients. Typically their TOTAL WBC yield is 4-5000. That's it. They set
up three 3 color tubes to look at CD3, CD14, and 3
chemokines.(cytometer= Facscan). The end result- they collect MAYBE
1000 total events/tube, get (on average) 100 monocyte events. Chemokine
receptor expression is usually a SHIFT, not a clearly separated
population. I guess I could feel better about analyzing 100 monocytes
for chemokine expression if positives were very +/+ (quadrant2). But
since you are looking at- what, 25-50 events, shifting across a marker;
this just really makes me wonder. If you pick up ANY noise in your
line- do you not worry? Is it because the FSC/SSC is so far over (from
usual debris) for mono's that the gate can't possibly have erroneous
events? My worry was even 10 "odd events" could inflate quadrant3 (-/-),
thus affecting the percentage profiles in Quad's 1/2/4.

Am I a worry-wort, or is this a legitimate concern? I also thought a
slide (confocal or something along those lines) would make more sense
than flow. (Thank you to everyone that sugessted confocal or CompuCyte LSC).

I have since heard from several people who run bacteria and collect only
100-500 events and say their data is very tight. Is this because they
have a very bright, very specific signal?

To those that suggested additional markers: they cannot add any more
(control)markers, because that would decrease the data collection even
more. (damned if you do/damned if you don't!)

Thanks again everyone-



Bunny






*******************************************************
Bunny Cotleur +*+ Bunny Cotleur
Cleveland Clinic Foundation *+* 2001 Lester RD
Neurosciences NC30 +*+ Valley City, OH 44280
9500 Euclid Avenue *+* 330-483-4800
Cleveland, OH 44195 +*+ bunny at cotleur.com
216-444-1164 *+*
cotleua at ccf.org +*+

*******************************************************
When you do something, you should burn yourself completely, like a good
bonfire, leaving no trace of yourself.
(Shunryu Suzuki)


From hms at shapirolab.com Tue Apr 11 17:35:02 2000
From: hms at shapirolab.com (Howard Shapiro)
Date: Tue, 11 Apr 2000 18:35:02 -0400
Subject: analysis of few cells
In-Reply-To: <38F24CC3.A3D636E2@cotleur.com>
Message-ID: <4.2.0.58.20000411181733.009e16d0@shell1.shore.net>


Bunny Cotleur writes-


>I wonder if I could gather some opinions on releveance as it relates to
>minimum numbers of events collected.
>I am assisting a lab in developing FACS analysis of PBMC's and monocytes
>in CSF. They informed me that when they run FACS, they are "lucky" to
>get 100 events. I raised the questions of relevance, stating that those
>100 "events" could as easily contain some non-cell events as cell
>events. They insist their data is good becasue it compares somewhat (?)
>with a lab in Europe they are collaborating with. We have had many
>conversations on this.
>(I should mention- neither lab has direct FACS experience. They got all
>their protocols second hand, and just collect what they collect).
>Additional information: they are looking at chemokine expression
>(another difficult task) on these few cells- and I believe they are
>using the PBMC's to set up isotype controls. I doubt they even use comp
>controls.
>
>Please throw your 2cents my way. And if any of you do this type of
>analysis (few cells) with alternative techniques- I'd love to hear about it!

Attallah et al (Attallah AM, Yeatman TJ, Noguchi PD, Johnson JB:
Antibody-dependent cell-mediated cytotoxicity: detection by automated flow
cytometry with ultramicro techniques. Science 1980; 209:404-6) found, using
microsyringe pumps for sample delivery, that antibody-dependent
cell-mediated cytotoxicity could be measured by analyzing as few as 1000
leukocytes. With the volumes and delivery systems used on conventional
instruments, I'd worry, as Bunny does, about how real the 100 events her
colleagues measure might be. This problem is tailor-made for the CompuCyte
LSC, although, if you don't have access to one, it might be possible to
hook the appropriate fluidics up to, say, a FACScan and analyze very small
numbers of cells.

-Howard





From millere at icrf.icnet.uk Wed Apr 12 02:49:08 2000
From: millere at icrf.icnet.uk (Eric Miller)
Date: Wed, 12 Apr 2000 08:49:08 +0100 (BST)
Subject: annexin V stain questions
In-Reply-To: <s8f1f36d.072@gateway.hsc.wvu.edu>
Message-ID: <Pine.SGI.3.96.1000412084606.10963704A-100000@seagull.lif.icnet.uk>


There is one very good reason why the unbound stain causes no problems:
threshold. If you are thresholding on FSC then it is highly unlikely that
any unbound dye molecules will be large enough to trigger a pulse. Not
washing out unbound stain does feature in many protocols, such as
Vindelov's DNA protocol.

Eric P Miller
Edinburgh Medical Oncology Unit

"Everyone can be correct, all at the same time. That's the
thing about quantum."
Terry Pratchett, Lords and ladies

On Mon, 10 Apr 2000, Jamie Brewer wrote:

>
> I am writing to inquire about an annexin V stain I am used today from Pharmingen. After
> calling the techinical support service as Pharmingen, I was given this E-mail
> address to correspond to with my unanswered question. The confusion comes in that
> the Pharmingen protocol does not have me wash off any unbound annexin prior to flow
> cytometry analysis. This is completely contradictory to any fluorescent stain I
have ever
> used. Typically, the unbound stain fluoresces when analyzed, losing any specificity the
> system had. My results seemed to turn out as expected today but I don't understand why
> the unbound stain is not causing me a problem. I have been unable to find any reference
> to the annexin being "active" only in the bound form as opposed to the unbound form
> or any statement indicating the something in the binding buffer inactivates unbound
> annexin. The technician at Pharmingen didn't have any idea why the unbound stain is
> not causing a problem. Do you have any understanding of this? Also, the protocol does
> not offer any option to fix the cells, something which I really need to do since I
> am using potentially infectious human cells in a common flow cytometry facility. The
> Pharmingen representative said that she knew of some people who had fixed the cells
> and it worked alright but not as well as if the cells were not fixed. Do you have
> any advice/suggestions?
>
> Please correspond to:
> Jamie Brewer
> jbrewer at hsc.wvu.edu
> Microbiology/Immunology Department, Mary Babb Randolph Cancer Center, West Virginia
> University
>



From CJett at Compucyte.com Tue Apr 11 15:23:07 2000
From: CJett at Compucyte.com (CJett)
Date: Tue, 11 Apr 2000 16:23:07 -0400
Subject: analysis of few cells
Message-ID: <F32AEE0410D3D311AF110090278C8A32066670@COMPMAIL>


I do human Monocytes all day here, In most of my studies a 100ul wb blood
sample can easily yield 1000 mono's in about 7-10 min from a lysed sample
using a FSC trigger.
for a whole blood sample ( no lyse) using a flour trigger instead of the
FSC and dilute 1/20 (to reduce coincidence) and you can get 1000 in about 10
min on LOW flow rate....

A 100 event file will contain about 2% noise ( more if whole blood)..this
drops after 300 events...statistically around 1000 is a safe bet.

-----Original Message-----
From: bunny [mailto:bunny at cotleur.com]
Sent: Monday, April 10, 2000 5:51 PM
To: cyto-inbox
Subject: analysis of few cells



Fellow Flowers:
I wonder if I could gather some opinions on releveance as it relates to
minimum numbers of events collected.
I am assisting a lab in developing FACS analysis of PBMC's and monocytes
in CSF. They informed me that when they run FACS, they are "lucky" to
get 100 events. I raised the questions of relevance, stating that those
100 "events" could as easily contain some non-cell events as cell
events. They insist their data is good becasue it compares somewhat (?)
with a lab in Europe they are collaborating with. We have had many
conversations on this.
(I should mention- neither lab has direct FACS experience. They got all
their protocols second hand, and just collect what they collect).
Additional information: they are looking at chemokine expression
(another difficult task) on these few cells- and I believe they are
using the PBMC's to set up isotype controls. I doubt they even use comp
controls.

Please throw your 2cents my way. And if any of you do this type of
analysis (few cells) with alternative techniques- I'd love to hear about it!
Thanks-
--



Bunny






*******************************************************
Bunny Cotleur +*+ Bunny Cotleur
Cleveland Clinic Foundation *+* 2001 Lester RD
Neurosciences NC30 +*+ Valley City, OH 44280
9500 Euclid Avenue *+* 330-483-4800
Cleveland, OH 44195 +*+ bunny at cotleur.com
216-444-1164 *+*
cotleua at ccf.org +*+

*******************************************************
When you do something, you should burn yourself completely, like a good
bonfire, leaving no trace of yourself.
(Shunryu Suzuki)


From Rudolf.Braun at med.uni-jena.de Wed Apr 12 07:44:09 2000
From: Rudolf.Braun at med.uni-jena.de (Rudolf Braun)
Date: Wed, 12 Apr 2000 14:44:09 +0200
Subject: analysis of few cells
Message-ID: <s8f48bd8.020@mail-gate.med.uni-jena.de>


Bunny,
as a reference for low frequency events look at the publication in J.Immunol.Methods
212: 89-98 (1998); Detection of antigen-specific T cell cytokine expression in whole
blood by flow cytometry; Suni, M.A., Picker, L.J., Maino, V.C.

I hope this helps
Ruedi

<<< bunny <bunny at cotleur.com> 4/11 11:31p >>>

Fellow Flowers:
I wonder if I could gather some opinions on releveance as it relates to
minimum numbers of events collected.
I am assisting a lab in developing FACS analysis of PBMC's and monocytes
in CSF. They informed me that when they run FACS, they are "lucky" to
get 100 events. I raised the questions of relevance, stating that those
100 "events" could as easily contain some non-cell events as cell
events. They insist their data is good becasue it compares somewhat (?)
with a lab in Europe they are collaborating with. We have had many
conversations on this.
(I should mention- neither lab has direct FACS experience. They got all
their protocols second hand, and just collect what they collect).
Additional information: they are looking at chemokine expression
(another difficult task) on these few cells- and I believe they are
using the PBMC's to set up isotype controls. I doubt they even use comp controls.

Please throw your 2cents my way. And if any of you do this type of
analysis (few cells) with alternative techniques- I'd love to hear about it!
Thanks-
--



Bunny






*******************************************************
Bunny Cotleur +*+ Bunny Cotleur
Cleveland Clinic Foundation *+* 2001 Lester RD
Neurosciences NC30 +*+ Valley City, OH 44280
9500 Euclid Avenue *+* 330-483-4800
Cleveland, OH 44195 +*+ bunny at cotleur.com
216-444-1164 *+*
cotleua at ccf.org +*+

*******************************************************
When you do something, you should burn yourself completely, like a good
bonfire, leaving no trace of yourself.
(Shunryu Suzuki)




From hms at shapirolab.com Tue Apr 11 16:40:48 2000
From: hms at shapirolab.com (Howard Shapiro)
Date: Tue, 11 Apr 2000 17:40:48 -0400
Subject: Vital-staining
In-Reply-To: <s8f1b90d.037@SV-BK-02.PEI.DE>
Message-ID: <4.2.0.58.20000411172525.009e26b0@shell1.shore.net>


Renate Wenig writes-



>I want to count absolute cell-number of vital cells with FACS.
>Until now I try to stain the dead cells with PI but it doesn't work:
>After detaching cells from the flaks bottom with EDTA all cells are PI
>positiv in FACS.
>Can anybody give me useful tips or instructions for vital-staining of
>adherent cells?

Many of the PI-positive cells you see after detachment are probably dead;
it is possible that all of them are. Can you plate them after you detach
them, which would establish viability?

The problem here is that procedures used to detach cells from flasks may
permeabilize the membrane, transiently or permanently. If the
permeabilization is permanent, the cells are dead; if transient, they can
recover. If you don't need to keep the cells viable (as for sorting) after
the flow cytometric analysis, but only want to know the fraction or number
of cells which were viable while they were in the flask, you should use a
staining procedure before detaching the cells. There are several which
would work; you might try 10-25 ug/mL 7-aminoactinomycin D
(7-AAD)(excitation 488 nm, emission 675 nm). This will only get into the
dead cells. Then, wash the cells and detach them, with 10-25 ug/ml plain
actinomycin D added to all wash solutions and to the final suspending
medium. Actinomycin D is nonfluorescent, and will bind to DNA in the cells
which are permeabilized by the detachment and other preparative procedures;
the cells which were originally dead will contain much more 7-AAD than
those which were not, so you will know what the viable fraction was before
you started abusing the cells. This trick is based on work by Terry
Fetterhoff et al and Ingrid Schmid et al, among others; there are other
techniques you could use, but this is what I'd try first.

-Howard


From M.Cooley at unsw.EDU.AU Wed Apr 12 03:58:18 2000
From: M.Cooley at unsw.EDU.AU (Margaret Cooley)
Date: Wed, 12 Apr 2000 18:58:18 +1000
Subject: Immunology Conference in Sydney, Australia December 2000
Message-ID: <200004120901.TAA08670@sam.comms.unsw.EDU.AU>

Dear Flowers:

This may be of interest to some.

30th Annual Conference of the
Australasian Society for Immunology
University of New South Wales, Sydney, Australia
10-15 December 2000

A major focus of the conference will be the regulation of the immune system
and the interactions between innate and adaptive immunity. Specific topics
will include inflammation, antigen presentation, immune cell development,
lymphocyte activation, autoimmunity, responses to infection and parasites,
and vaccine development.

Invited speakers :
Douglas Fearon, Stephen Galli, Lewis Lanier, Charles Mackay,
Fabienne Mackay, Polly Matzinger, Arthur Weiss, Jack Gauldie, Stephen
Ullrick, Pedro Romero, Johnathan Sprent

A Tumour Immunology Workshop will be held on Dec 14-15.

Further information is available on www.asi2000.org

or contact:
AIDA Corporate Events
PO Box 1472
Bondi Junction NSW 1355
Australia
Fax +61 2 9567 9912
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From M.G.Kelly at queens-belfast.ac.uk Wed Apr 12 03:34:47 2000
From: M.G.Kelly at queens-belfast.ac.uk (Martin Kelly)
Date: Wed, 12 Apr 2000 09:34:47 +0100 (GMT Daylight Time)
Subject: annexin V stain questions
In-Reply-To: <s8f1f36d.072@gateway.hsc.wvu.edu>
Message-ID: <SIMEON.10004120947.A@eagle.fujin.qub.ac.uk>


Dear Jamie,
as a clinician working along with a colleague with expertise in
flow cytometry, and having had some experience in this field, I
will make my humble contribution to this question!
We work with sputum and bronchoalveolar lavage cells, staining
with both annexin V (AV) and propidium iodide (PI). We had been
concerned r.e. exactly the same issue, and particularly, were
concerned that there was not an extra wash stage for the PI. My
colleague with the previous flow cytometry experience would
concur exactly with what you have said. In practice, we do wash
after labelling. Anecdotally, I can say that we have not found
much difference. We did run one experiment where we ran a sample
where we had done an extra wash, and compared with those where
we didn't wash. There seemed to be no difference in staining in
this n=1 experiment.
I seem to remember reading somewhere that washing labelling may
affect the cell membrane permeability, so affecting PI uptake
(and possibly also AV staining), tho' we haven't really seemed
to see this in practice. I will see if I can find where I got
this information, and see if I can firm up my 'vague
recollection'!
I know this doesn't really answer even the first half of your
question, but I am interested to see that someone else has had
the same thoughts on this. I would be very interested to hear
what comes of any discussion, so that we can improve our
practice too, if necessary.
I am surprised that Pharmingen weren't able to offer help...

Best wishes,

Martin Kelly


On Mon, 10 Apr 2000 15:29:22 -0400 Jamie Brewer
<jbrewer at hsc.wvu.edu> wrote:

>
> I am writing to inquire about an annexin V stain I am used today from Pharmingen. After
> calling the techinical support service as Pharmingen, I was given this E-mail
> address to correspond to with my unanswered question. The confusion comes in that
> the Pharmingen protocol does not have me wash off any unbound annexin prior to flow
> cytometry analysis. This is completely contradictory to any fluorescent stain I
have ever
> used. Typically, the unbound stain fluoresces when analyzed, losing any specificity the
> system had. My results seemed to turn out as expected today but I don't understand why
> the unbound stain is not causing me a problem. I have been unable to find any reference
> to the annexin being "active" only in the bound form as opposed to the unbound form
> or any statement indicating the something in the binding buffer inactivates unbound
> annexin. The technician at Pharmingen didn't have any idea why the unbound stain is
> not causing a problem. Do you have any understanding of this? Also, the protocol does
> not offer any option to fix the cells, something which I really need to do since I
> am using potentially infectious human cells in a common flow cytometry facility. The
> Pharmingen representative said that she knew of some people who had fixed the cells
> and it worked alright but not as well as if the cells were not fixed. Do you have
> any advice/suggestions?
>
> Please correspond to:
> Jamie Brewer
> jbrewer at hsc.wvu.edu
> Microbiology/Immunology Department, Mary Babb Randolph Cancer Center, West Virginia
> University

----------------------
Martin Kelly
Department of Clinical Biochemistry,
Institute of Clinical Science,
Grosvenor Road,
BELFAST, BT12 6BJ. UK.
Tel No: +44 (0) 28 9026 3267
Fax No: +44 (0) 28 9023 6543




From thomas at tritechinc.com Tue Apr 11 15:48:10 2000
From: thomas at tritechinc.com (Joanne Thomas)
Date: Tue, 11 Apr 2000 16:48:10 -0400
Subject: CD4/CD8 coexpression in pb
Message-ID: <003c01bfa3f7$3ffa96a0$1801a8c0@jill.tritechinc.com>

Andrea:
We have found this phenomena to be associated with a protein in the plasma of certain patients. If you wash the cells once before staining it actually goes away. Didn't believe it myself until I tried the before and after! I don't think its associated with a pathological process as some of our normal control have showed this reversible co-expression as well. Give it a try!
Joanne


Joanne Thomas, M.S.
Director of Operations
TRITECH Field Engineering
2014 Renard Court, Suite I
Annapolis, MD 21401
1-800-886-7004 (USA)
1-410-266-1522
410-266-0935 (FAX)
thomas at tritechinc.com
-----Original Message-----
From: Andrea Illingworth <dcdsflow at mint.net>
To: Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu>
Date: Tuesday, April 11, 2000 4:19 PM
Subject: CD4/CD8 coexpression in pb


We have noticed occasional cases with a fair amount of CD4/CD8 coexpression in peripheral bloods (up to 10% coexpression within the lymphoid gate). Most of these cases show no CMC or morphological abnormalities and we are not sure about the clinical significance of this findings. Does anybody know a "non-malignant" explanation for this finding? QC of these antibodies looks fine.

Thanks for your help!

Andrea J. Illingworth, M.S. (ASCP)
Dahl-Chase Diagnostic Services
Flow Cytometry
333 State Street
Bangor, Maine 04401

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From pif at box-p.nih.gov Wed Apr 12 07:19:13 2000
From: pif at box-p.nih.gov (Paula Fukushima)
Date: Wed, 12 Apr 2000 08:19:13 -0400
Subject: DVD-RAM for data storage
In-Reply-To: <4.2.2.20000410110258.01025420@facstaff.wisc.edu>
Message-ID: <KIEAJECBELBBKLLLOFFFIEEDCAAA.pif@box-p.nih.gov>


Hi Justin,
I use a Jazz drive. It is inexpensive and holds 2.2GB data per disk.
The wave of the future, though, is to store data on the Web.

Paula

-----Original Message-----
From: Janet E. Lewis [mailto:jelewis1 at facstaff.wisc.edu]
Sent: Monday, April 10, 2000 12:19 PM
To: cyto-inbox
Subject: Re: DVD-RAM for data storage



Justin,

At 03:52 PM 4/6/00 -0500, thus did you send forth:
>We are in the process of switching to a larger and faster data server
>(Windows NT) to store our flow cytometry data. We will be using either one
>or two 9GB drives. I am wrestling with long term archive options that
>currently include 4mm DAT tape, CD-RW/CD-R or DVD-RAM. I am leaning toward
>DVD-RAM because of the 5.2GB capacity and longevity of the media. The down
>side is that DVD-RAM is not a mature technology like CD-RW/CD-R or 4mm DAT.
> We'll probably install it on a fast MAC in the lab (the server is in
>another building) and use Retrospect backup software so that we can retain
>Cellquest file attributes without Filetyping when we pull archived files
>from the disk. The last threads in the flow e-mail archives relating to
>DVD are dated 1997. Has anyone used this storage technology since then?
>Thanks.

We have Retrospect (which I like), but you don't need to use Retrospect to
preserve the file attributes. Just enable Services for Macintosh on NT
Server. I have a CD burner on a Mac and I copy the flow data directly from
the NT Server shared "Mac" disk (using Chooser) onto a local hard disk (for
burning speed), then burn them using the Mac Folders and Files format in
Adaptec Toast.

DAT tapes don't have the longevity of either CD or DVD media - only a few
months for certain, and it's a real pain (and time-consuming) to hunt down
individual files for restoration. The advantage of CD-R is that if need
be, any Mac can read them. Plus, since it is write-once media, it makes it
more acceptable as an official "archive of record." DVD is indeed a
wonderful thing, and I myself lust for a DVD-writer, but until they've
adopted a reasonably stable format, I'm holding off switching to that
system for archives. I think in a year or so, they'll have hammered
something out.

--Janet


----------------------------------------
Janet E. Lewis
Computer Support (PC & Mac)
UWCCC Flow Cytometry Facility
UWCCC Imaging Facility
Clinical Sciences Center, K4/535
600 Highland Ave.
Madison, WI 53792-5666

(608)263-0313
(608)263-9226
jelewis1 at facstaff.wisc.edu
----------------------------------------




From oberyszyn.2 at osu.edu Tue Apr 11 07:55:46 2000
From: oberyszyn.2 at osu.edu (Andrew Oberyszyn)
Date: Tue, 11 Apr 2000 08:55:46 -0400
Subject: OSU 3rd Annual Spring Cytometry Symposium
Message-ID: <4.2.0.58.20000411084736.009aaa20@pop.service.ohio-state.edu>

3rd Annual Spring Cytometry Symposium
Thursday May 4th, 2000

HOSTED BY:
The Ohio State University Comprehensive Cancer Center
Analytical Cytometry Shared Resource Laboratory

LOCATION:
Medical Heritage Center
Prior Health Sciences Library
376 West 10th Avenue
Columbus, OH 43210

Day of Events:
8:30AM Welcome and Opening Remarks
Fredika Robertson, Ph.D
Professor/Director of the Ohio State University
Comprehensive Cancer Center
Analytical Cytometry Shared Resource Laboratory

9:45AM Visualizing Early Antigen T-Cell Responses to Oral MBP In Vivo
Richard M. Wardrop, III
Medical Scientist Program Fellow,
The Ohio State University Department of Molecular
Virology, Immunology, and Molecular Genetics

10:15AM Recent Advances in Multiparametric Cell Cycle Analysis
James W. Jaccobberger, Ph.D
Associate Professor, Director Flow Cytometry
Case Western Reserve University Comprehensive Cancer
Center

10:45AM Sensitivity and Specificity of Tetramer Technology
Albert Donnenberg, Ph.D
Associate Professor of Medicine & Director of the
University of Pittsburgh Cancer
Institute Flow Cytometry Facility

11:30AM Sorting Chromosomes Using Flow Cytometry
Dr. Nigel P. Carter
The Sanger Centre
Wellcome Trust Genome Campus
Hinxton Cambridge CB10 1SA UK

Sponsored By: Beckman-Coulter

Call 800-338-8830 X8764 for further information

Refreshments will be provided
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(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)
Andy Oberyszyn, M.S.
The Ohio State University
Analytical Cytometry Laboratory
416 Comprehensive Cancer Center
410 West 12th Avenue
Columbus, Ohio 43210
Tel: 614/292-FLOW(3569)
Fax: 614/292-7335
E-Mail: cytometry at osu.edu
Web Page:
<http://cytometry.med.ohio-state.edu>http://cytometry.med.ohio-state.edu
(-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-)

"What if the Hokey Pokey is really what it's all about?!?"

From TSAWYER at MCO.EDU Thu Apr 13 08:53:04 2000
From: TSAWYER at MCO.EDU (Tom Sawyer)
Date: Thu, 13 Apr 2000 09:53:04 -0400
Subject: CD4/CD8 COEXPRESSION
Message-ID: <s8f5990b.050@at851.mco.edu>


Greetings Andrea, The simplest explanation for a relatively low percentage of
"unexpected" double positive events is aggregates. Most folks are very aware of the
problems of aggregates or coincident events in DNA analysis but tend to forget about
them when doing immunophenotyping.

Good Luck,

Tom Sawyer
MCO Flow Cytometry Laboratory



From MHughes at picr.man.ac.uk Thu Apr 13 08:57:02 2000
From: MHughes at picr.man.ac.uk (Michael Hughes)
Date: Thu, 13 Apr 2000 14:57:02 +0100
Subject: murine megakaryocyte labelling
Message-ID: <c=US%a=_%p=Paterson_Institu%l=LANCELOT-000413135702Z-23844@lancelot.picr.man.ac.uk>


Can anyone recommend specific antibody labeling for murine megakaryocyte?

Mike Hughes


From oes at rsp.is Thu Apr 13 05:42:02 2000
From: oes at rsp.is (oes@rsp.is)
Date: Thu, 13 Apr 2000 10:42:02 +0000
Subject: Cell surface staining and ethanol fixation
Message-ID: <38F5A47A.3C76802@rsp.is>


hi there

I would like to know wether it is is ok to stain cells for cell surface
markers fix them with ethanol keep them in a freezer for one month and
then stain for DNA content using PI. What effect would that have on my
cell surface markers in the Flowcytometry to fix the cells in ethanol

Hope some one will respond a.s.a.p.

yours

?lafur E Sigurj?nsson
oes at rsp.is




From peter.kierulf at ioks.uio.no Thu Apr 13 07:59:18 2000
From: peter.kierulf at ioks.uio.no (peter.kierulf@ioks.uio.no)
Date: Thu, 13 Apr 2000 14:59:18 +0200
Subject: No subject
Message-ID: <v04210102b51b6f9ed11e@[129.240.42.244]>

Dear Flowers

So many precious answers from this group - thus this question:

We have for some time looked into apoptosis using among other parameters
Annexin - V. Generally this works well using A-V-FITC from Pharmingen. We now
want to bind onto A-V (non-FITC-tagged) bound to PS, the mouse monoclonal
antibody Mab 3184 Chemicon clone RUU-WAC2A (designed for ELISA or immunohistochemistry).
So far we have had no success as evidenced from binding a PE tagged antimouse
antibody (rabbit) to the PS-A-V-Mab3184 complex.

We have searched for other A-V antibodies, but all suppliers appear to use the same clone, RUU-WAC2A.

We sure appreciate all comments / suggestions.


Thanks.


Peter Kierulf
e-mail: peter.kierulf at ioks.uio.no



From Alice.L.Givan at Dartmouth.EDU Wed Apr 12 18:42:43 2000
From: Alice.L.Givan at Dartmouth.EDU (Alice L. Givan)
Date: 12 Apr 2000 19:42:43 EDT
Subject: macho competition
Message-ID: <9836376@cupid.Dartmouth.EDU>


Hello Flowers,
I need some information for my current writing and teaching projects: What are the
greatest number of parameters that exist on a current, operational flow cytometer?
If you think you might have a winner, send me the specs (number of lasers, number of
fluorescence parameters, number of scatter parameters, etc) and I will compile the
results and send them back to the network. Needless to say, no prizes (other than
bragging rights).

Alice

Alice L. Givan
Englert Cell Analysis Laboratory
of the Norris Cotton Cancer Center
Dartmouth Medical School
Lebanon, New Hampshire NH 03756
tel 603-650-7661
fax 603-650-6130
givan at dartmouth.edu



From stighe at zoo.uvm.edu Wed Apr 12 15:55:18 2000
From: stighe at zoo.uvm.edu (Scott Tighe)
Date: Wed, 12 Apr 2000 16:55:18 -0400
Subject: PI deactivation
Message-ID: <38F4E2B6.86646AB9@zoo.uvm.edu>



Is anyone aware of a simple method to deal with PI. We do a lot of DNA
cell cycle analysis and I would like to have a method to clean pipet
handles, keyboard,ect... I'm hoping that reacting with some type of
agent to possibly make it safer? any help would be great.

Scott Tighe
Flow Cytometry Core Facility
Vermont cancer Ctr
A214 Medical Alumni
Burlington, Vermont 0540


From a.pizzey at ucl.ac.uk Thu Apr 13 06:08:51 2000
From: a.pizzey at ucl.ac.uk (Arnold Pizzey)
Date: Thu, 13 Apr 2000 12:08:51 +0100
Subject: mouse myeloperoxidase
Message-ID: <3.0.6.32.20000413120851.008cf420@pop-server.bcc.ac.uk>


Greetings all,


Does anyone know of a company which markets an anti-mouse myeloperoxidase?
preferably flurochrome conjugated but I'll take unconjugated.

all the search results on the mail archive seem to be for anti-human.


best wishes to all


Arnold

_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/
Arnold Richard Pizzey
Department of Haematology
Royal Free and University College London Medical School
98 Chenies Mews
London WC1E 6HX
U.K

voice: +44 0207-209-6234
Fax: +44 0207-209-6222
email: a.pizzey at ucl.ac.uk
_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/


From thomas.nebe at ikc.ma.uni-heidelberg.de Thu Apr 13 05:05:36 2000
From: thomas.nebe at ikc.ma.uni-heidelberg.de (Nebe, Thomas C.)
Date: Thu, 13 Apr 2000 12:05:36 +0200
Subject: CSF / few cells
Message-ID: <7021552C81F1D111B6FF006097AA2312C23471@intrant.kli-ma>


First work was done by Prof. Dr. T.O. Kleine in Marburg 10 yrs ago. His
protocol is published in the book "Durchflusszytometire in der klinischen
Zelldiagnostik", Schattauer, Stuttgart.
It is a no wash no lyse method on CSF using LDS 751 to gate on nucleated
cells. The problem of analysing few cells is discussed. He established
normal values for CSF.
There is a working group in Germany on CSF and flow operated by Dr. Mix in
Rostock (eilhard.mix at med.uni-rostock.de).
Thomas Nebe

Dr.med. C. Thomas Nebe
Universitaetsklinikum Mannheim
Zentrallabor
Theodor-Kutzer-Ufer 1-3
D-68167 Mannheim
Tel. +49 621 383-3485
FAX +49 621 383-73 3485
+49 621 383-3819
e-mail: thomas.nebe at ikc.ma.uni-heidelberg.de

Bitte besuchen Sie unsere informativen Webseiten unter
http://www.ma.uni-heidelberg.de/inst/ikc/ (Internet)
http://pandora/inst/ikc/ (Intranet)



From waitumbi at net2000ke.com Thu Apr 13 19:39:14 2000
From: waitumbi at net2000ke.com (John Waitumbi)
Date: Thu, 13 Apr 2000 17:39:14 -0700
Subject: Quantification of macrophage phagocytosis
References: <01BFA3D3.BBB39A40@bootp-62-199.bootp.Virginia.EDU>
Message-ID: <00ef01bfa5aa$1d952440$5e569ed4@w000>


Raymond

Try quenching FITC-fluorescence of extracellular bacteria by addition of
trypan blue or crystal violet (trypan blue 1 mg/ml or crystal violet 0.8
mg/ml at pH = 7.4). Blut (1984) 49: 315-323. Remember to flush the machine
with bleach.

John

------------------------------------------------------------------------
Dr. John N. Waitumbi
Walter Reed Project
P.O. Box 30137 Nairobi
Kenya
Phone +254 35 22942
Email:waitumbi at net2000ke.com
Fax +254 35 22903
Electronic Fax Service: 1-603- 947-4913
----- Original Message -----
From: D Raymond <dpr4w at virginia.edu>
To: cyto-inbox
Sent: Tuesday, April 11, 2000 7:33 AM
Subject: Quantification of macrophage phagocytosis


>
> I am trying to quantify macrophage bacterial phagocytosis but have had
numerous problems.
> I am trying to quantify the phagocytosis of E. coli by the murine
macrophage cell line
> RAW 264.7. I have tried using FITC labelled E. coli but have had problems
quenching
> external bacteria. The RAW cells take up the quenching agent (ethidium
bromide) almost
> immediately They do appear viable when I quantify them by tryphan blue
exclusion and,
> under similar conditions, we see significant cytokine production. If
anyone has any
> experience with this technique I would appreciate any input. Thank you
for your time.
> -Daniel Raymond (Charlottesville, VA)
>



From klk1 at CDC.GOV Wed Apr 12 15:52:51 2000
From: klk1 at CDC.GOV (Kellar, Kathryn L. (Kathi))
Date: Wed, 12 Apr 2000 16:52:51 -0400
Subject: Labtek Dishes problem
Message-ID: <1537895B1173D1118D9300A0246212B6035DB6D3@MCDC-ATL-4>


DITTO!! I cannot tell you how useful I had found their product in the past.

Kathi Kellar, PhD
National Center for Infectious Diseases
CDC
1600 Clifton Rd, NE
Atlanta, GA 30333
404-639-1419
FAX 404-639-3129
klk1 at cdc.gov



-----Original Message-----
From: jpr at flowcyt.cyto.purdue.edu
[mailto:jpr at flowcyt.cyto.purdue.edu]
Sent: Tuesday, April 11, 2000 4:20 PM
To: Cytometry Mailing List
Subject: Labtek Dishes problem


Colleagues
We have been using Labtek (made by Nunc) Chambered
Coverglass dishes (8 chambers) for several years (#136439).
[see a
picture on
http://www.cyto.purdue.edu/flowcyt/confocal/cfcl7.htm

We found out recently that they have redesigned all their
plates, by
making small (but significant physical size changes) They
also
changed the glass bases - bottom line is the new dishes
don't fit
any of our specially designed heatsinks, holders, etc. They
also
stuck a "handle" on top of the clear plastic lis, so now you
cant
image through the dishes. Real smart people must have just
taken
over at Labtek!!!!

So, if anyone has some of the old dishes (sterile packs of
course) in
stock, we love to hear from you, we will be happy to
exchange equal
value, give up a graduate student or two, or a few bottles
of nice
plonk.....whatever it takes.

If anyone out there has any influence with Nunc, I think
they pretty
much destroyed a really successful product....!!!

HELP please....
thanks
Paul Robinson

J.Paul Robinson, PhD PH:(765)4940757
Professor of Immunopharmacology
Professor of Biomedical Engineering
Purdue University FAX:(765)4940517
EMAIL:jpr at flowcyt.cyto.purdue.edu
WEB: http://www.cyto.purdue.edu


From flow at post.queensu.ca Wed Apr 12 16:02:36 2000
From: flow at post.queensu.ca (Derek Schulze)
Date: Wed, 12 Apr 2000 14:02:36 -0700
Subject: annexin V stain questions
In-Reply-To: <s8f1f36d.072@gateway.hsc.wvu.edu>
Message-ID: <3.0.3.32.20000412140236.007790e8@post.queensu.ca>


We always wash our cells after incubation with Annexin V. I suspect that
you will get a better signal to noise response (better separation between
positive and negative) if you wash the cells. It may, however, not be as
significant as one might think.

A light fixation with paraformaldehyde will render the cells cooked with
membranes intact. I would suggest a starting point of 1% for 5 minutes. I
will assume you are already using PI to gate out (count) necrotic cells,
which will have leaky membranes. If you see this number as being higher
than an unfixed control then you will have to adjust your fixation
procedure accordingly. Remember, you can also keep increasing the
percentage of paraformaldehyde and use the above procedure to assess if you
are affecting the membranes (increasing PI+). The higher the
paraformaldehyde the greater your peace of mind.

Hy humble opinion.


At 03:29 PM 2000-04-10 -0400, Jamie Brewer wrote:
>
>I am writing to inquire about an annexin V stain I am used today from
Pharmingen. After
>calling the techinical support service as Pharmingen, I was given this E-mail
>address to correspond to with my unanswered question. The confusion comes
in that
>the Pharmingen protocol does not have me wash off any unbound annexin
prior to flow
>cytometry analysis. This is completely contradictory to any fluorescent
stain I have ever
>used. Typically, the unbound stain fluoresces when analyzed, losing any
specificity the
>system had. My results seemed to turn out as expected today but I don't
understand why
>the unbound stain is not causing me a problem. I have been unable to find
any reference
>to the annexin being "active" only in the bound form as opposed to the
unbound form
>or any statement indicating the something in the binding buffer
inactivates unbound
>annexin. The technician at Pharmingen didn't have any idea why the unbound
stain is
>not causing a problem. Do you have any understanding of this? Also, the
protocol does
>not offer any option to fix the cells, something which I really need to do
since I
>am using potentially infectious human cells in a common flow cytometry
facility. The
>Pharmingen representative said that she knew of some people who had fixed
the cells
>and it worked alright but not as well as if the cells were not fixed. Do
you have
>any advice/suggestions?
>
>Please correspond to:
>Jamie Brewer
>jbrewer at hsc.wvu.edu
>Microbiology/Immunology Department, Mary Babb Randolph Cancer Center, West
Virginia
>University
>
>
- Derek Schulze

Flow Cytometry and Confocal Microscopy Core Facility
Cancer Research Labs
Queen's University
Kingston, Ontario
Canada


From vannacci at pharm.unifi.it Thu Apr 13 05:15:47 2000
From: vannacci at pharm.unifi.it (Alfredo Vannacci)
Date: Thu, 13 Apr 2000 12:15:47 +0200
Subject: No subject
Message-ID: <000001bfa531$3ce6c340$9745d996@pharm.unifi.it>

Dear Flowers,
I would like to know if someone has experience in gating human basophils
according to their FS/SS pattern.
I read some articles in wich was described a gate on lymphocyte region.
Any comment would be apreciated.

Dr Alfredo Vannacci, MD

Universit? degli Studi di Firenze
Dipartimento di Farmacologia Preclinica e Clinica "Mario Aiazzi Mancini"
Unit? Operativa di Tossicologia Medica e Medicina delle
Farmacotossicodipendenze
Viale Pieraccini 6, 50139, Firenze, Italia

e-mail vannacci at pharm.unifi.it

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From l.barber at pmci.unimelb.edu.au Thu Apr 13 00:44:42 2000
From: l.barber at pmci.unimelb.edu.au (Barber, Lesley (PMC))
Date: Thu, 13 Apr 2000 15:44:42 +1000
Subject: Vital-staining
Message-ID: <58983447B50ED311954300A0C98A352501498058@PMC-MAIL01>


In addition to Howard's comments.
I use citric saline to detach my cells as it is less harsh than EDTA or
trypsin and the cells reattach well.. I find I get excellent viability
staining with this method.
Recipe:
For a 10x stock solution on citric saline,
1.35M potassium chloride
0.15M sodium citrate
Autoclave and store at 4C, dilute 1:10 with sterile distilled water before
use.
Remove all media, flood flask bottom with 1x citric saline (prewarmed to
37C) incubate at 37C for maximum 5 minutes. Tap flask bottom to gently
detach all cells. Decant cells, mix well for single cell suspension and add
equal volumes PBS. Centrifuge and wash with fresh PBS.
Cheers Lesley
Mrs. Lesley Barber
Edith Margaret Collie Centre for Blood Cell Therapies
Div. Haematology/Medical Oncology
Peter MacCallum Cancer Institute
East Melbourne, 3002
VICTORIA , AUSTRALIA
Tel 61 3 9656 1957
Fax 61 3 9656 1811


> -----Original Message-----
> From: Howard Shapiro [SMTP:hms at shapirolab.com]
> Sent: Wednesday, April 12, 2000 7:41 AM
> To: Cytometry Mailing List
> Subject: Re: Vital-staining
>
>
> Renate Wenig writes-
>
>
>
> >I want to count absolute cell-number of vital cells with FACS.
> >Until now I try to stain the dead cells with PI but it doesn't work:
> >After detaching cells from the flaks bottom with EDTA all cells are PI
> >positiv in FACS.
> >Can anybody give me useful tips or instructions for vital-staining of
> >adherent cells?
>
> Many of the PI-positive cells you see after detachment are probably dead;
> it is possible that all of them are. Can you plate them after you detach
> them, which would establish viability?
>
> The problem here is that procedures used to detach cells from flasks may
> permeabilize the membrane, transiently or permanently. If the
> permeabilization is permanent, the cells are dead; if transient, they can
> recover. If you don't need to keep the cells viable (as for sorting)
> after
> the flow cytometric analysis, but only want to know the fraction or number
> of cells which were viable while they were in the flask, you should use a
> staining procedure before detaching the cells. There are several which
> would work; you might try 10-25 ug/mL 7-aminoactinomycin D
> (7-AAD)(excitation 488 nm, emission 675 nm). This will only get into the
> dead cells. Then, wash the cells and detach them, with 10-25 ug/ml plain
> actinomycin D added to all wash solutions and to the final suspending
> medium. Actinomycin D is nonfluorescent, and will bind to DNA in the
> cells
> which are permeabilized by the detachment and other preparative
> procedures;
> the cells which were originally dead will contain much more 7-AAD than
> those which were not, so you will know what the viable fraction was before
> you started abusing the cells. This trick is based on work by Terry
> Fetterhoff et al and Ingrid Schmid et al, among others; there are other
> techniques you could use, but this is what I'd try first.
>
> -Howard


From jose.diaz_romero at pharma.Novartis.com Thu Apr 13 03:31:51 2000
From: jose.diaz_romero at pharma.Novartis.com (jose.diaz_romero@pharma.Novartis.com)
Date: Thu, 13 Apr 2000 09:31:51 +0100
Subject: CD4/CD8 coexpression in pb
Message-ID: <412568C0.002EDE1E.00@nts1.novartis.com>


In peripheral blood, according to the classical scheme of T-cell
differentiation, mature CD4+ and CD8+ T-cells are mutually exclusive subsets.
However, mature T-cells with double-positive phenotype (CD4+/CD8+) can be found
in very small numbers (1-3%) in human, and rat peripheral blood, and in a higher
percentage, up to 15%, in rhesus monkeys (Macaca mulatta), cynomolgus monkeys (
Macaca fascicularis) and African green monkeys (Cercophitecus aethiops). In
normal adult pigs, a substantial proportion (10-60%) of peripheral lymphocytes
can simultaneously express CD4 and CD8. Coexpression of CD4 and CD8 can be
generated in peripheral blood T-cells by treatment with lectin which induces CD8
a biosynthesis and cell surface expression. The CD4+/CD8+ population derives
from CD4+/CD8- T-cells that become double positive upon activation.



Rabinowitz, R., R. Hadar, and M. Schlesinger. 1997. The appearance of the
CD4+CD8+phenotype on activated T cells: possible role of antigen transfer.
Hum.Immunol. 55:1.


Morris, D.L. and W.J. Komocsar. 1997. Immunophenotyping analysis of
peripheral blood, splenic, and thymic lymphocytes in male and female rats.
J.Pharmacol.Toxicol.Methods 37:37.


Zuckermann, F.A. and R.J. Husmann. 1996. Functional and phenotypic analysis
of porcine peripheral blood CD4/CD8 double-positive T cells. Immunology 87:500.

Currier, J.R., Stevenson, K.S., Kehn, P.J., Zheng, K., Hirsch, V.M. and
Robinson, M.A. (1999) Contributions of CD4+, CD8+, and CD4+CD8+ T cells to
skewing within the peripheral T cell receptor beta chain repertoire of healthy
macaques. Hum.Immunol. 60:209.

Ramirez, F., A.J. McKnight, A. Silva, and D. Mason. 1992. Glucocorticoids
induce the expression of CD8 alpha chains on concanavalin A-activated rat CD4+ T
cells: induction is inhibited by rat recombinant interleukin 4. J.Exp.Med. 176
:1551.


O'Donovan, M.R., S. Johns, and P. Wilcox. 1995. The effect of PHA stimulation
on lymphocyte sub-populations in whole- blood cultures. Mutagenesis 10:371.


Paliard, X., R.W. Malefijt, J.E. de Vries, and H. Spits. 1988. Interleukin-4
mediates CD8 induction on human CD4+ T-cell clones. Nature 335:642.




Elevated levels of peripheral double-positive cells have previously been
reported in one apparently normal adult male, and this phenotype appear to be
increased in a number of clinical conditions, including myasthenia gravis,
multiple sclerosis, idiopathic thrombocytopenic purpura, Behcet?s syndrome,
HIV-infection, inflammatory bowel disease, Kawaski disease, lepramatous leprae,
and patients suffering some types of neoplasia.


Kay, N.E., N. Bone, M. Hupke, and A.P. Dalmasso. 1990. Expansion of a
lymphocyte population co-expressing T4 (CD4) and T8 (CD8) antigens in the
peripheral blood of a normal adult male. Blood 75:2024.

Hirao, J. and K. Sugita. 1998. Circulating CD4+CD8+ T lymphocytes in
patients with Kawasaki disease. Clin.Exp.Immunol. 111:397.

Senju, M., K.C. Wu, Y.R. Mahida, and D.P. Jewell. 1991. Coexpression of
CD4 and CD8 on peripheral blood T cells and lamina propria T cells in
inflammatory bowel disease by two colour immunofluorescence and flow cytometric
analysis. Gut 32:918.

Mizuki, M., S. Tagawa, T. Machii, M. Shibano, E. Tatsumi, K. Tsubaki, H.
Tako, A. Yokohama, S. Satou, J. Nojima, T. Hirota, and T. Kitani. 1998.
Phenotypical heterogeneity of CD4+CD8+ double-positive chronic T lymphoid
leukemia. Leukemia 12:499.

Ottenhoff, T.H., D.G. Elferink, P.R. Klatser, and R.R. de Vries. 1986.
Cloned suppressor T cells from a lepromatous leprosy patient suppress
Mycobacterium leprae reactive helper T cells. Nature 322:462.

Weiss, L., A. Roux, S. Garcia, C. Demouchy, N. Haeffner-Cavaillon, M.D.
Kazatchkine, and M.L. Gougeon. 1998. Persistent expansion, in a human
immunodeficiency virus-infected person, of V beta-restricted CD4+CD8+ T
lymphocytes that express cytotoxicity-associated molecules and are committed to
produce interferon-gamma and tumor necrosis factor-alpha. J.Infect.Dis.
178:1158.

Watanabe, N., S.C. De Rosa, A. Cmelak, R. Hoppe, L.A. Herzenberg, and M.
Roederer. 1997. Long-term depletion of naive T cells in patients treated for
Hodgkin's disease. Blood 90:3662.

Senju, M., K.C. Wu, Y.R. Mahida, and D.P. Jewell. 1991. Coexpression of
CD4 and CD8 on peripheral blood T cells and lamina propria T cells in
inflammatory bowel disease by two colour immunofluorescence and flow cytometric
analysis. Gut 32:918.



Jose Diaz Romero
Transplantation Deparment
Novartis Pharma AG
Basel
Switzerland





From stetler at box-s.nih.gov Thu Apr 13 08:56:05 2000
From: stetler at box-s.nih.gov (Maryalice Stetler-Stevenson)
Date: Thu, 13 Apr 2000 09:56:05 -0400
Subject: Fwd: RE: analysis of few cells
Message-ID: <v04220801b51b80d9ce82@[156.40.224.68]>


For the application being discussed you are correct and I do
feel better when there are 1000 events to study. However, in studying
lymphoid tumors using a multiparameter approach we can make an
accurate diagnosis with 100 cells- no problem. If a patient with
hairy cell leukemia, with surface lambda light chin expression,
bright CD22, bright CD11c, and negative kappa, if you identify 100
cells with this immunophenotype I all it diagnostic. You just can't
pick a number for all applications.

Maryalice


>
>I do human Monocytes all day here, In most of my studies a 100ul wb blood
>sample can easily yield 1000 mono's in about 7-10 min from a lysed sample
>using a FSC trigger.
>for a whole blood sample ( no lyse) using a flour trigger instead of the
>FSC and dilute 1/20 (to reduce coincidence) and you can get 1000 in about 10
>min on LOW flow rate....
>
>A 100 event file will contain about 2% noise ( more if whole blood)..this
>drops after 300 events...statistically around 1000 is a safe bet.
>
>-----Original Message-----
>From: bunny [mailto:bunny at cotleur.com]
>Sent: Monday, April 10, 2000 5:51 PM
>To: cyto-inbox
>Subject: analysis of few cells
>
>
>
>Fellow Flowers:
>I wonder if I could gather some opinions on releveance as it relates to
>minimum numbers of events collected.
>I am assisting a lab in developing FACS analysis of PBMC's and monocytes
>in CSF. They informed me that when they run FACS, they are "lucky" to
>get 100 events. I raised the questions of relevance, stating that those
>100 "events" could as easily contain some non-cell events as cell
>events. They insist their data is good becasue it compares somewhat (?)
>with a lab in Europe they are collaborating with. We have had many
>conversations on this.
>(I should mention- neither lab has direct FACS experience. They got all
>their protocols second hand, and just collect what they collect).
>Additional information: they are looking at chemokine expression
>(another difficult task) on these few cells- and I believe they are
>using the PBMC's to set up isotype controls. I doubt they even use comp
>controls.
>
>Please throw your 2cents my way. And if any of you do this type of
>analysis (few cells) with alternative techniques- I'd love to hear about it!
>Thanks-
>--
>
>
>
>Bunny
>
>
>
>
>
>
>*******************************************************
>Bunny Cotleur +*+ Bunny Cotleur
>Cleveland Clinic Foundation *+* 2001 Lester RD
>Neurosciences NC30 +*+ Valley City, OH 44280
>9500 Euclid Avenue *+* 330-483-4800
>Cleveland, OH 44195 +*+ bunny at cotleur.com
>216-444-1164 *+*
>cotleua at ccf.org +*+
>
>*******************************************************
>When you do something, you should burn yourself completely, like a good
>bonfire, leaving no trace of yourself.
>(Shunryu Suzuki)

Maryalice Stetler-Stevenson
Director Flow Cytometry Unit
Laboratory of Pathology, NCI, NIH


From Grant.Howes at coulter.com Wed Apr 12 12:49:47 2000
From: Grant.Howes at coulter.com (Grant.Howes@coulter.com)
Date: Wed, 12 Apr 2000 13:49:47 -0400
Subject: Cytometry Companies and the Internet
Message-ID: <852568BF.0061AD08.00@ctcffan6.coulter.com>


As I work for one of the Cytometry Companies, I would not normally respond to
the bulletin board. However, as you raise the issue of software upgrades, I
thought I should mention that one of our software packages, EXPO2 software
(build 320), is available for download from the following address:

www.appliedcytometry.com


With best regards,

Grant Howes
Marketing Manager, Cytometry Instrument Systems,
Beckman Coulter, Miami




From TSAWYER at MCO.EDU Thu Apr 13 15:31:33 2000
From: TSAWYER at MCO.EDU (Tom Sawyer)
Date: Thu, 13 Apr 2000 16:31:33 -0400
Subject: Fwd: Re: RE: CD4/CD8 COEXPRESSION
Message-ID: <s8f5f66e.026@at851.mco.edu>

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From BarrenP at MedImmune.com Fri Apr 14 07:34:52 2000
From: BarrenP at MedImmune.com (Barren, Phil)
Date: Fri, 14 Apr 2000 08:34:52 -0400
Subject: Companies that Lease Lab Equipment
Message-ID: <B8483A460B7CD211908B00A0C969C02C1CAE1C@MEDIMMUNE4>


FLOWers:

Does anyone happen to know of a company that leases Lab equipment??

Tks

Pb


From rr5845 at hotmail.com Thu Apr 13 19:41:43 2000
From: rr5845 at hotmail.com (Raj Nathan)
Date: Thu, 13 Apr 2000 17:41:43 PDT
Subject: CD33/CD38/CD34 Staining on slides
Message-ID: <20000414004143.87110.qmail@hotmail.com>


Hi
I want to stain cells on slides for CD34/CD33and CD38 possibly double
staining or even triple staining. I want to use antibodies tagged with
flourocent labels like FITC,PE etc. Is it possible to do a double or even
triple staining on slides if anyone can help me with suggetions or
publications or even suggesting antibodies that are manufactured for such
work i will be very gratefull.
Thanks
Raj.
______________________________________________________
Get Your Private, Free Email at http://www.hotmail.com



From Sciex at AOL.COM Wed Apr 12 14:42:10 2000
From: Sciex at AOL.COM (Sciex@AOL.COM)
Date: Wed, 12 Apr 2000 15:42:10 EDT
Subject: Antibody to mouse P21 or P27
Message-ID: <90.2ebd19d.26262b92@aol.com>



In a message dated 4/12/00 1:52:01 PM,
helix.mgh.harvard.edu at helix.mgh.harvard.edu writes:

<<
I am looking for an antibody either fluorochrome conjugated or not
specific for mouse P21 or P27. Please let me know if this is available.

David M. Dombkowski
dombkowski at helix.mgh.harvard.edu
Flow Cytometry-Pathology-CNY rm7017
149 13th Street
Massachusetts General Hospital-East
Boston, MA 02129
Tel. (617)726-1683
Fax.(617)724-3164 >>

Exalpha Biologicals, Inc. (800.395.1137) has an anti mouse/rat p27 antibody
cat no. C155M - applications include western blot, immunoprecipitation, and
immunohistochemistry.
Regards,
John Castracane
Exalpha Biologicals, Inc.


From wittrup at MIT.EDU Thu Apr 13 12:49:26 2000
From: wittrup at MIT.EDU (Dane Wittrup)
Date: Thu, 13 Apr 2000 13:49:26 -0400
Subject: UV (280 nm) excitation in flow for lanthanides?
Message-ID: <v03020900b51bb8d0fc51@[18.63.2.76]>


Does anybody have their flow instrument set up for excitation from 280-300
nm? We'd like to excite a lanthanide bound in calmodulin, using Trp as the
donor to the ion.

_____________________________________
K. Dane Wittrup
J.R. Mares Professor
Chemical Engineering & Bioengineering
Massachusetts Institute of Technology
Bldg 66-552
77 Massachusetts Ave.
Cambridge, MA 02139
PH 617-253-4578 FAX 617-258-5766




From Zsolt.Juranyi at sanofi-synthelabo.com Fri Apr 14 03:05:47 2000
From: Zsolt.Juranyi at sanofi-synthelabo.com (Zsolt Juranyi)
Date: Fri, 14 Apr 2000 9:05:47 +0100
Subject: unknown population from rat blood
Message-ID: <"000414065814Z.WT18429. 1*/PN=Zsolt.Juranyi/O=FRSANOFI/PRMD=SANOFI/ADMD=ATLAS/C=FR/"@MHS>


Hello everybody, please help me.
Flow cytometric analysis of rat blood (after haemolysis) shows an unknown population
which appears in low FS (close by lymphocytes) and from low SS to high SS channels
. These cells (?) are negative for all simple AB (45/3/4/8/11/45R) but they represent
a huge part of the sample. Are they just simple debris might originate from not perfect
haemolysis ?
Zsolt Juranyi
Sanofi-Synthelabo


From KHOLMES at niaid.nih.gov Wed Apr 12 10:51:56 2000
From: KHOLMES at niaid.nih.gov (Kevin Holmes)
Date: Wed, 12 Apr 2000 11:51:56 -0400
Subject: tandem conjugates
Message-ID: <0CF057718479D111AEFA00A0C989933005702CB7@atlas.niaid.nih.gov>


Marty,
The reference I have for the first fluorescent tandem conjugates is:
Glazer and Stryer , Biophys. J. 43:383-386, 1983,
They made PE-APC tandems.
Kevin
Kevin L. Holmes, Ph.D.
Chief, Flow Cytometry Section
Research Technologies Branch
Bldg. 7, Room 01
NIAID, NIH

Phone: 301-496-9071
FAX: 301-402-4532
Email: kholmes at .niaid.nih.gov



-----Original Message-----
From: Marty Bigos [mailto:bigos at stanford.edu]
Sent: Monday, April 10, 2000 5:26 PM
To: cyto-inbox
Subject: tandem conjugates



Can someone tell me who (and when) developed the first energy
transfer tandem conjugate fluorochrome? References would be helpful
as well. Thanks.

Marty Bigos
Operations Manager
Stanford Shared FACS Facility
bigos at stanford.edu
(voice) 650-723-6959
(fax) 650-725-8564


From kbahjat at ufl.edu Thu Apr 13 16:00:16 2000
From: kbahjat at ufl.edu (Keith Bahjat)
Date: Thu, 13 Apr 2000 17:00:16 -0400
Subject: PI deactivation
In-Reply-To: <38F4E2B6.86646AB9@zoo.uvm.edu>
Message-ID: <B51BAD9F.B59%kbahjat@ufl.edu>


How dangerous is a membrane impermeable dye?? Unless it can resurrect dead
epithelial cells, it should not have access to the DNA of living cells, and
thus shouldn't be much of a safety hazard.

We always wipe everything down with ethanol, but I've yet to see data
showing membrane impermeable DNA intercalating dyes cause problems for
living cells. I think the MSDS claims are based on a general fear of
anything that has the word "DNA" in it.

Anyone have data to contradict this??

kb

--
Keith Bahjat
Graduate Assistant
University of Florida
College of Medicine
Gainesville, Florida
Voice: (352) 392-4887
Fax: (352) 392-5393
e-mail: kbahjat at ufl.edu


> From: Scott Tighe <stighe at zoo.uvm.edu>
> Organization: UVM VCC
> Date: Wed, 12 Apr 2000 16:55:18 -0400
> To: Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu>
> Subject: PI deactivation
>
>
>
> Is anyone aware of a simple method to deal with PI. We do a lot of DNA
> cell cycle analysis and I would like to have a method to clean pipet
> handles, keyboard,ect... I'm hoping that reacting with some type of
> agent to possibly make it safer? any help would be great.
>
> Scott Tighe
> Flow Cytometry Core Facility
> Vermont cancer Ctr
> A214 Medical Alumni
> Burlington, Vermont 0540



From CPRUSSIN at niaid.nih.gov Thu Apr 13 16:38:39 2000
From: CPRUSSIN at niaid.nih.gov (Calman Prussin)
Date: Thu, 13 Apr 2000 17:38:39 -0400
Subject: gating human basophils
Message-ID: <0CF057718479D111AEFA00A0C9899330049EFBD6@atlas.niaid.nih.gov>


When we have analyzed PFA fixed PBMC, the basophils (IgE+, lin-) cells were
somewhat between the lymphs and moncytes on the FSC/SSC plot. I have not
read it, but Larry Schwartz has a paper purifying basophils by sorting on
their scatter characteristics [Kepley et. al, J. Imm Methods 175:1 (1990)].

Calman
> ----------
> From: Alfredo Vannacci
> Sent: Thursday, April 13, 2000 6:15 AM
> To: Cytometry Mailing List
>
> Dear Flowers,
> I would like to know if someone has experience in gating human basophils
> according to their FS/SS pattern.
> I read some articles in wich was described a gate on lymphocyte region.
> Any comment would be apreciated.
>
> Dr Alfredo Vannacci, MD
>
> Universit? degli Studi di Firenze
> Dipartimento di Farmacologia Preclinica e Clinica "Mario Aiazzi Mancini"
> Unit? Operativa di Tossicologia Medica e Medicina delle
> Farmacotossicodipendenze
> Viale Pieraccini 6, 50139, Firenze, Italia
>
> e-mail vannacci at pharm.unifi.it
>
>


From virginia.litwin at bms.com Thu Apr 13 11:59:30 2000
From: virginia.litwin at bms.com (Virginia M Litwin)
Date: Thu, 13 Apr 2000 12:59:30 -0400
Subject: superoxide and myeloperoxidase activity in neutrophils
Message-ID: <38F5FCF2.6D4EF278@bms.com>

Dear Flowers:

Thanks very much to those who sent the extremely useful replies
to my request for elastase activity in neutrophils. Now I am
wondering if anyone knows of any flow methods to measure
superoxide and myeloperoxidase activity in neutrophils.
Any comments are appreciated.

Virginia
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From virginia.litwin at bms.com Wed Apr 12 11:10:25 2000
From: virginia.litwin at bms.com (Virginia M Litwin)
Date: Wed, 12 Apr 2000 12:10:25 -0400
Subject: rsCD4
References: <8439A208B822D311B4DD0008C7EAAA0B019BE6C8@phsexch12.partners.org>
Message-ID: <38F49FF1.F2F579AB@bms.com>

Dear Doug:

Progenics Pharmaceuticals Inc. makes sCD4, which is now available through the
NIH AIDS reagent bank.
http://www.aidsreagent.org/

Virginia





"Olson, Douglas" wrote:

> Does anyone know where to get recombinant soluble CD4 (to be used for in
> vitro competitive binding assays)? Biogen used to make it but it seems they
> don't anymore.
>
> Thanx,
> do
>
> -----------------------------------
> Douglas Olson
> Experimental Hematology/AIDS Research Center
> Massachusetts General Hospital
> Harvard Medical School
> 149 13th Street
> Boston, MA 02129
> (617)724-2668 - Phone
> (617)726-4691 - Fax
> Dolson4 at partners.org
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From oamfilho at cpqrr.fiocruz.br Wed Apr 12 13:00:40 2000
From: oamfilho at cpqrr.fiocruz.br (Olindo Assis)
Date: Wed, 12 Apr 2000 15:00:40 -0300
Subject: unexpected T and B phenotypes after culture
References: <3.0.1.32.20000410114821.00824e30@popmail.ucsd.edu>
Message-ID: <000801bfa4a9$0450dda0$380983c8@cpqrr.fiocruz.br>


We have been phenotyping splenocytes from different strains of mouse before
and after stimulation with bacteria (H. pylori) antigens. We are basically
labeling the cells using anti-CD4, anti-CD8 and anti-CD45(B220) PE from
SIGMA. Labeling procedures were done in separated tubes.
Frequently we have noticed that the sum of CD4 + CD45 + CD8 cells from the
unstimulated cultures give us a number over 100%.
It is interesting to observe that this phenomena is observed only in
unstumulated cells. Cultures in the presence of H. pylori antigens does not
show this problem. Moreover it was observed for all mouse strains evaluated
independent of the age. We have used Balb/C, C3H/HeN and C57/BL6.
It is interesting to observe that these overestimation is observed only in
the region corresponding to larger cells. We first suggested that this could
be due to the presence of doublets of CD4 and CD45 cells. However it could
be also due to the presence of cells co-expressing both phenotypes.
In order to solve this we are currently evaluating the presence of double
labeled cells using anti-CD4 FITC and anti-CD45 PE in the same tube.
However, if we find double labeled cells we still have the question whether
they are representing doublets or bi-phenotypic cells.
Does any one have experience of phenotypic analysis of T and B splenocytes
after control cultures? Any suggestions?
Is that possible that CD45 (B220) could be a marker for cultured CD4 cells?


Any help is appreciated,

Olindo




From rafaeln at vetvir.unizh.ch Fri Apr 14 03:02:17 2000
From: rafaeln at vetvir.unizh.ch (Rafael Nunez)
Date: Fri, 14 Apr 2000 09:02:17 +0100
Subject: Fwd: RE: analysis of few cells
In-Reply-To: <v04220801b51b80d9ce82@[156.40.224.68]>
Message-ID: <l03130301b51c7ba8c778@[130.60.97.37]>


I will said that for some samples like CSF, is pretty normal to have very
low number of cells, actually is normal to find less than 10 cells/mm3.
However, to make diagnosis with low number of cells is a completely
different ball game. At that point is very important to have very well set
up your staining technique and your setting of the equipment should be very
well defined. Moreover, you can also should define that your procedures
are very reproducible and be sure that there is not chance that the
multicolor analysis has compensation problems (pretty often with three
colors). Then you can said that your results represent the real situation
of the CSF of your patient.
Recently there is a growing number of publications, mainly due to the
tetramers technology, that define as significant some numbers like 0.005%
for a sample of 200.000 cells (J of Imm and J Exp Med). That means about
100 cells having this marker. I found a little bit disturbing to jump from
this type of in vitro test to the bed of patients and believe that 100
cells for this type of assays is significant. Therefore, reporting that
100 cells in the CSF of a patient is also significant. I just remind to
some of the nonmedical community here, that to get a CSF sample without any
possibility of contaminating blood is a real feat and not too many profis
can boast of having a reproducible tap techniques without blood
contamination. You do not need too much, just enought in the walls of the
needle and your small population is obscure for the other cells coming from
the blood.
I hope that my comments will be helpful to you.
Regards Rafael

\|/
(o o)
________________________________oOo__(_)__oOo_________________________________
___/\_ | Rafael Nunez mailto:rafaeln at vetvir.unizh.ch
/ o \/| | University Inst.for Virology http://www.vetvir.unizh.ch/
/ _| | Winterthurerstr. 266a Telephone: (+41) 1 6358710
/_/\__/-\/ | 8057 Zurich SWITZERLAND Faximile : (+41) 1 6358911
______________________________________________________________________________









From kbahjat at ufl.edu Thu Apr 13 16:48:22 2000
From: kbahjat at ufl.edu (Keith Bahjat)
Date: Thu, 13 Apr 2000 17:48:22 -0400
Subject: Quantification of macrophage phagocytosis
In-Reply-To: <00ef01bfa5aa$1d952440$5e569ed4@w000>
Message-ID: <B51BB8E5.B61%kbahjat@ufl.edu>


When quantifying phagocytosis, rather than quenching extracellularly bound
signal, we use a control tube that is incubated on ice. Any fluorescence in
these cells should be due to surface bound particles (whether it is e. coli,
dextran, or ova), and this can be compared to the 37 degree sample.

kb

--
Keith Bahjat
Graduate Assistant
University of Florida
College of Medicine
Gainesville, Florida
Voice: (352) 392-4887
Fax: (352) 392-5393
e-mail: kbahjat at ufl.edu


> From: "John Waitumbi" <waitumbi at net2000ke.com>
> Organization: Walter Reed Project
> Reply-To: "John Waitumbi" <waitumbi at net2000ke.com>
> Date: Thu, 13 Apr 2000 17:39:14 -0700
> To: Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu>
> Subject: Re: Quantification of macrophage phagocytosis
>
>
> Raymond
>
> Try quenching FITC-fluorescence of extracellular bacteria by addition of
> trypan blue or crystal violet (trypan blue 1 mg/ml or crystal violet 0.8
> mg/ml at pH = 7.4). Blut (1984) 49: 315-323. Remember to flush the machine
> with bleach.
>
> John
>
> ------------------------------------------------------------------------
> Dr. John N. Waitumbi
> Walter Reed Project
> P.O. Box 30137 Nairobi
> Kenya
> Phone +254 35 22942
> Email:waitumbi at net2000ke.com
> Fax +254 35 22903
> Electronic Fax Service: 1-603- 947-4913
> ----- Original Message -----
> From: D Raymond <dpr4w at virginia.edu>
> To: cyto-inbox
> Sent: Tuesday, April 11, 2000 7:33 AM
> Subject: Quantification of macrophage phagocytosis
>
>
>>
>> I am trying to quantify macrophage bacterial phagocytosis but have had
> numerous problems.
>> I am trying to quantify the phagocytosis of E. coli by the murine
> macrophage cell line
>> RAW 264.7. I have tried using FITC labelled E. coli but have had problems
> quenching
>> external bacteria. The RAW cells take up the quenching agent (ethidium
> bromide) almost
>> immediately They do appear viable when I quantify them by tryphan blue
> exclusion and,
>> under similar conditions, we see significant cytokine production. If
> anyone has any
>> experience with this technique I would appreciate any input. Thank you
> for your time.
>> -Daniel Raymond (Charlottesville, VA)
>>
>



From daviesd2 at icrf.icnet.uk Thu Apr 13 09:54:42 2000
From: daviesd2 at icrf.icnet.uk (Derek Davies)
Date: Thu, 13 Apr 2000 15:54:42 +0100 (BST)
Subject: annexin V stain questions
In-Reply-To: <s8f1f36d.072@gateway.hsc.wvu.edu>
Message-ID: <Pine.SGI.3.96.1000413154048.96717C-100000@seagull.lif.icnet.uk>



Hi Jamie,

I certainly dont wash my samples after the annexin binding step - there
may be an slightly increased background due to non-specific sticking of
the annexin to negative cells but the true positives will still be
obvious. It is best to avoid any unnecessary centrifugation steps
as it may lead to differential cell loss of the apoptotic cells. Unbound
annexin (or any antibody) will not cause a problem as it will be too
small to trigger the machine.

Regarding fixation, you presumably want to fix the cells prior to any
staining? The trouble with this is you will lose the ability to separate
the early apoptotic (FITC+/PI-) from the late (FITC+/PI+) ones. The time
when the cells are passing through that early stage in an individual
cell is quite short so it is better to avoid this! If you really have to
fix, then you are limiting your choices to methods that can be performed
on fixed cells eg subG1, TUNEL unless you use the 7AAD/AD technique to
identify the dead cells prior to fixing and annexin staining.

Derek


On Mon, 10 Apr 2000, Jamie Brewer wrote:
> I am writing to inquire about an annexin V stain I am used today from Pharmingen. After
> calling the techinical support service as Pharmingen, I was given this E-mail
> address to correspond to with my unanswered question. The confusion comes in that
> the Pharmingen protocol does not have me wash off any unbound annexin prior to flow
> cytometry analysis. This is completely contradictory to any fluorescent stain I
have ever
> used. Typically, the unbound stain fluoresces when analyzed, losing any specificity the
> system had. My results seemed to turn out as expected today but I don't understand why
> the unbound stain is not causing me a problem. I have been unable to find any reference
> to the annexin being "active" only in the bound form as opposed to the unbound form
> or any statement indicating the something in the binding buffer inactivates unbound
> annexin. The technician at Pharmingen didn't have any idea why the unbound stain is
> not causing a problem. Do you have any understanding of this? Also, the protocol does
> not offer any option to fix the cells, something which I really need to do since I
> am using potentially infectious human cells in a common flow cytometry facility. The
> Pharmingen representative said that she knew of some people who had fixed the cells
> and it worked alright but not as well as if the cells were not fixed. Do you have
> any advice/suggestions?
>
> Please correspond to:
> Jamie Brewer
> jbrewer at hsc.wvu.edu
> Microbiology/Immunology Department, Mary Babb Randolph Cancer Center, West Virginia
> University
>

************************************************************************
Derek Davies Voice: (44) 0207 269 3394
FACS Laboratory, FAX: (44) 0207 269 3100
Imperial Cancer Research Fund, e_mail: derek.davies at icrf.icnet.uk
London, UK mobile: 07790 604112

Web Page: http://www.icnet.uk/axp/facs/davies/index.html

In tenebris lux
*************************************************************************





From joy.mundy at imvs.sa.gov.au Thu Apr 13 19:45:33 2000
From: joy.mundy at imvs.sa.gov.au (Joy Mundy)
Date: Fri, 14 Apr 2000 10:15:33 +0930
Subject: Sorting CD4+CD3+ cells
Message-ID: <000301bfa5aa$bde6b4e0$f767140a@ihi31382.imvs.sa.gov.au>


Hi everybody,

I am passing the following message on behalf of a researcher in our HIV
research labs. In our lab we are familiar with the analysis of T cell
subsets, currently using the Coulter tetraCHROME reagent. However this
researcher is interested in cell sorting and how to set up the BD flow
sorter to get the cells of interest. As a consequence I am of no use
whatsoever. The antibodies that we currently have at our disposal are the
Coulter tetraCHROME CD45 FITC/CD4 PE/CD8 ECD/CD3 PC5 and BD CD3/CD8/CD4
together with the BD Tritest control. Analysing the cells are no problem
it's just how th set up the sorter to get the correct cells out!

This is the problem;

Basically what I want to do is as follows
Take 40mls patient blood, ficoll separation.
Stain PBMCs in such a way that I can put them thru a cell sorter and
collect as many cells as possible as need to extract DNA from the
following , to then do a series of PCR reactions to determine integrated
HIV DNA.
Interested in CD4+ lymphocytes (CD3+/CD4+), CD8+ and the small number of
double negative cells (cd4-/CD8-) as these are possibly CD4+ cells that
have down regulated their receptor post HIV infection. I guess the ideal
scenario is to pass the cells thru and collect all CD3+ cells and then
the rest. Then pass thru these CD3+ cells and differentiate between the
4s and the 8s????

Thanks any suggestions welcome !

Joy Mundy
Division of Human Immunology
I.M.V.S.
PO Box 14
Rundle Mall PO
Adelaide 5000
Australia

joy.mundy at imvs.sa.gov.au

Phone (08) 8222-3476
Fax (08) 8232-4092



From Roederer at drmr.com Thu Apr 13 15:42:51 2000
From: Roederer at drmr.com (Mario Roederer)
Date: Thu, 13 Apr 2000 13:42:51 -0700
Subject: CD4/CD8 coexpression in pb
In-Reply-To: <412568C0.002EDE1E.00@nts1.novartis.com>
References: <412568C0.002EDE1E.00@nts1.novartis.com>
Message-ID: <v0422081cb51bdf23abdc@[128.218.108.229]>


I wanted to add to Jose's outstanding review of CD4+CD8+ ("DP") T
cells in peripheral blood. The level of CD8 expression (as well as
exactly the form, be it CD8 alpha/alpha or CD8 alpha/beta)
distinguishes different kinds of "DP" cells. As we showed in Blood
in 1997 [Watanabe et al], there are subsets of CD8dull CD4+ T cells
(that express CD8a/a homodimers) which can become prevalent in
certain diseases (such as late-stage AIDS, after chemotherapy), but
can be found in variable proportions in healthy adults. These cells
are unlikely to be CD4 cells that have upregulated CD8alpha, but
rather constitute a distinct lineage (because we can identify
differentiation & effector stages within the CD4+CD8dull, and have
determined the VBeta repertoire of the "naive" CD4+CD8-dull to be
very different from that of either CD4+ or CD8+ single-positive T
cells--and yes, I know that doesn't prove they're a distinct lineage,
but we can have that argument over a nice bottle of wine at ISAC).

In any case, there are also CD4+CD8+ which express CD8alpha/beta
heterodimers are are "bright" CD8. (These distinctions only work
well if you are using very bright CD8 reagents that have low
backgrounds). These "true" double positives are very rare, and
usually are a consequence of having dead cells: dead cells are
notoriously sticky. We showed that, especially after stimulation,
90% of "CD4+CD8+" cells are actually dead cells and could have nearly
any original phenotype. However, there are very rare "true" DP cells
that are not dead. Whether these are a distinct lineage of mature T
cell or whether they cells that have perhaps "spilled out" of the
thymus during development is not known to me.

Note that reports of CD4+CD8+ T cells making IL4 must be taken with a
grain of salt, because these are certainly dead cells. When
excluding dead cells after stimulation (by, e.g., EMA staining), the
few remaining true DP cells do not make IL4. Certain antibodies to
IL4 have a very high propensity to bind dead cells--although I
believe the particular clone we used that had this property has been
discontinued.

The finding of CD4+CD8+ in primates is likely not an artefact of dead
cells--there's simply too many to be explained by death. However, we
still need to determine if these cells are CD8-dull (i.e., CD8a/a) or
CD8-bright (and express CD8a/b heterodimers). This is important
because the CD8-dull cells (which can be either CD4+ or CD4-,
incidentally) are a very different "lineage" than the DP.

mr



From Sciex at aol.com Fri Apr 14 10:25:15 2000
From: Sciex at aol.com (Sciex@aol.com)
Date: Fri, 14 Apr 2000 11:25:15 EDT
Subject: CD4/CD8 coexpression in pb
Message-ID: <75.30954d8.2628925b@aol.com>


My two cents

We have found constant and significant numbers of CD4+/CD8+ cells in the pb
of normal individuals using the Lymphogram test (~1%, range 0.6 - 2.8%, n=
21). While it may be easier to explain these populations as background or
aggregates, I concure with Jose that this is a real population that can
fluxuate with various disorders.

see
Rapid and Simple Imunophenotypic characterization of Lymphocytes Using a New
Test. Bellido, M. et al. Haematologica 83: (8) pp 681-685. 1998.

Lymphogram has numberous advantages over conventional methods (for any one
interested see http://www.exalpha.com/lymphogram.htm for a complete
discription of the test and a comparison v. standard methods for CD4 and CD8
measurements).

Regards,
John Castracane
Exalpha Biologicals, Inc

In a message dated 4/13/00 3:19:41 PM, jose.diaz_romero at pharma.Novartis.com
writes:

<<

In peripheral blood, according to the classical scheme of
T-cell

differentiation, mature CD4+ and CD8+ T-cells are mutually exclusive
subsets.

However, mature T-cells with double-positive phenotype (CD4+/CD8+) can be
found

in very small numbers (1-3%) in human, and rat peripheral blood, and in a
higher

percentage, up to 15%, in rhesus monkeys (Macaca mulatta), cynomolgus
monkeys (

Macaca fascicularis) and African green monkeys (Cercophitecus aethiops).
In

normal adult pigs, a substantial proportion (10-60%) of peripheral
lymphocytes

can simultaneously express CD4 and CD8. Coexpression of CD4 and CD8 can
be

generated in peripheral blood T-cells by treatment with lectin which induces
CD8

a biosynthesis and cell surface expression. The CD4+/CD8+ population
derives

from CD4+/CD8- T-cells that become double positive upon activation.




Rabinowitz, R., R. Hadar, and M. Schlesinger. 1997. The appearance of
the

CD4+CD8+phenotype on activated T cells: possible role of antigen
transfer.

Hum.Immunol. 55:1.



Morris, D.L. and W.J. Komocsar. 1997. Immunophenotyping analysis
of

peripheral blood, splenic, and thymic lymphocytes in male and female
rats.

J.Pharmacol.Toxicol.Methods 37:37.



Zuckermann, F.A. and R.J. Husmann. 1996. Functional and phenotypic
analysis

of porcine peripheral blood CD4/CD8 double-positive T cells. Immunology
87:500.


Currier, J.R., Stevenson, K.S., Kehn, P.J., Zheng, K., Hirsch, V.M.
and

Robinson, M.A. (1999) Contributions of CD4+, CD8+, and CD4+CD8+ T cells
to

skewing within the peripheral T cell receptor beta chain repertoire of
healthy

macaques. Hum.Immunol. 60:209.


Ramirez, F., A.J. McKnight, A. Silva, and D. Mason. 1992.
Glucocorticoids

induce the expression of CD8 alpha chains on concanavalin A-activated rat
CD4+ T

cells: induction is inhibited by rat recombinant interleukin 4. J.Exp.Med.
176

:1551.



O'Donovan, M.R., S. Johns, and P. Wilcox. 1995. The effect of PHA
stimulation

on lymphocyte sub-populations in whole- blood cultures. Mutagenesis 10:371.



Paliard, X., R.W. Malefijt, J.E. de Vries, and H. Spits. 1988.
Interleukin-4

mediates CD8 induction on human CD4+ T-cell clones. Nature 335:642.





Elevated levels of peripheral double-positive cells have previously
been

reported in one apparently normal adult male, and this phenotype appear to
be

increased in a number of clinical conditions, including myasthenia
gravis,

multiple sclerosis, idiopathic thrombocytopenic purpura, Behcet?s
syndrome,

HIV-infection, inflammatory bowel disease, Kawaski disease, lepramatous
leprae,

and patients suffering some types of neoplasia.



Kay, N.E., N. Bone, M. Hupke, and A.P. Dalmasso. 1990. Expansion
of a

lymphocyte population co-expressing T4 (CD4) and T8 (CD8) antigens in
the

peripheral blood of a normal adult male. Blood 75:2024.


Hirao, J. and K. Sugita. 1998. Circulating CD4+CD8+ T lymphocytes
in

patients with Kawasaki disease. Clin.Exp.Immunol. 111:397.


Senju, M., K.C. Wu, Y.R. Mahida, and D.P. Jewell. 1991. Coexpression
of

CD4 and CD8 on peripheral blood T cells and lamina propria T cells
in

inflammatory bowel disease by two colour immunofluorescence and flow
cytometric

analysis. Gut 32:918.


Mizuki, M., S. Tagawa, T. Machii, M. Shibano, E. Tatsumi, K. Tsubaki,
H.

Tako, A. Yokohama, S. Satou, J. Nojima, T. Hirota, and T. Kitani.
1998.

Phenotypical heterogeneity of CD4+CD8+ double-positive chronic T
lymphoid

leukemia. Leukemia 12:499.


Ottenhoff, T.H., D.G. Elferink, P.R. Klatser, and R.R. de Vries.
1986.

Cloned suppressor T cells from a lepromatous leprosy patient
suppress

Mycobacterium leprae reactive helper T cells. Nature 322:462.


Weiss, L., A. Roux, S. Garcia, C. Demouchy, N. Haeffner-Cavaillon,
M.D.

Kazatchkine, and M.L. Gougeon. 1998. Persistent expansion, in a
human

immunodeficiency virus-infected person, of V beta-restricted CD4+CD8+
T

lymphocytes that express cytotoxicity-associated molecules and are committed
to

produce interferon-gamma and tumor necrosis factor-alpha.
J.Infect.Dis.

178:1158.


Watanabe, N., S.C. De Rosa, A. Cmelak, R. Hoppe, L.A. Herzenberg, and
M.

Roederer. 1997. Long-term depletion of naive T cells in patients treated
for

Hodgkin's disease. Blood 90:3662.


Senju, M., K.C. Wu, Y.R. Mahida, and D.P. Jewell. 1991. Coexpression
of

CD4 and CD8 on peripheral blood T cells and lamina propria T cells
in

inflammatory bowel disease by two colour immunofluorescence and flow
cytometric

analysis. Gut 32:918.




Jose Diaz Romero

Transplantation Deparment

Novartis Pharma AG

Basel

Switzerland

>>

-- End --


From preffer at helix.mgh.harvard.edu Thu Apr 13 22:14:44 2000
From: preffer at helix.mgh.harvard.edu (Frederic Preffer)
Date: Thu, 13 Apr 2000 23:14:44 -0400
Subject: analysis of few cells
In-Reply-To: <38F24CC3.A3D636E2@cotleur.com>
Message-ID: <200004131512.LAA0000002038@helix.mgh.harvard.edu>

Dear Bunny

I would be very cautious about looking at a few cells (<100); however i am
entirely confident that this can be done by flow cytometry.. we have looked at
hundreds of csf or vitreous fluids as clincal cases over the last few years.
When done by professional/ experienced folks like those i am happy to say work
in our lab here, the results are entirely trustworthy. The Compucytometer is
also likely able to do this type of work.

see
Kelliher AS, Lau S, Olszak I, Dombkowski DM and FI Preffer FI (1999)
Multiparameter Flow Cytometry on Cerebrospinal and Vitreous Fluid Specimens:
Use in Identifying Hematologic Malignancy in the Central Nervous System.
Cytometry 38: p336 #41. CAC Conference, Palm Springs CA

F Preffer


At 05:51 PM 4/10/00 -0400, bunny wrote:
>
>Fellow Flowers:
>I wonder if I could gather some opinions on releveance as it relates to
>minimum numbers of events collected.
>I am assisting a lab in developing FACS analysis of PBMC's and monocytes
>in CSF. They informed me that when they run FACS, they are "lucky" to
>get 100 events. I raised the questions of relevance, stating that those
>100 "events" could as easily contain some non-cell events as cell
>events. They insist their data is good becasue it compares somewhat (?)
>with a lab in Europe they are collaborating with. We have had many
>conversations on this.
>(I should mention- neither lab has direct FACS experience. They got all
>their protocols second hand, and just collect what they collect).
>Additional information: they are looking at chemokine expression
>(another difficult task) on these few cells- and I believe they are
>using the PBMC's to set up isotype controls. I doubt they even use comp
controls.
>
>Please throw your 2cents my way. And if any of you do this type of
>analysis (few cells) with alternative techniques- I'd love to hear about it!
>Thanks-
>--
>
>
>
>Bunny
>
>
>
>
>
>
>*******************************************************
>Bunny Cotleur +*+ Bunny Cotleur
>Cleveland Clinic Foundation *+* 2001 Lester RD
>Neurosciences NC30 +*+ Valley City, OH 44280
>9500 Euclid Avenue *+* 330-483-4800
>Cleveland, OH 44195 +*+ bunny at cotleur.com
>216-444-1164 *+*
>cotleua at ccf.org +*+
>
>*******************************************************
>When you do something, you should burn yourself completely, like a good
>bonfire, leaving no trace of yourself.
>(Shunryu Suzuki)
>
Frederic I. Preffer
Department of Pathology
Charlestown Navy Yard- 7140
Massachusetts General Hospital East
Charlestown, MA 02129

voice [617] 726-7481
fax [617] 724-3164
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From DOLSON4 at partners.org Fri Apr 14 11:33:02 2000
From: DOLSON4 at partners.org (Olson, Douglas)
Date: Fri, 14 Apr 2000 12:33:02 -0400
Subject:
Message-ID: <8439A208B822D311B4DD0008C7EAAA0B019BE6FB@phsexch12.partners.org>


Does anyone know of an antibody for TGF-beta suitable for flow?

thx,
do

----------------------------------------
Douglas Olson
Experimental Hematology/AIDS Research Center
Massachusetts General Hospital
Harvard Medical School
149 13th Street
Boston, MA 02129
(617)724-2668 - Phone
(617)726-4691 - Fax
Dolson4 at partners.org - Email




From behringer at mm11.ukl.uni-freiburg.de Sat Apr 15 12:23:07 2000
From: behringer at mm11.ukl.uni-freiburg.de (Dr. Dirk Behringer)
Date: Sat, 15 Apr 2000 18:23:07 +0100
Subject: Sorting of megakaryocytes?
Message-ID: <435D520BA7@mm11.ukl.uni-freiburg.de>


Dear flowers,

is there any easy to handle method to enrich megakaryocytes from bone
marrow by flow or magnetic bead methods?

Kind regards
Dirk
Dirk Behringer (behringer at mm11.ukl.uni-freiburg.de)
Medizin I, Studienzentrale, Universit„tsklinik Freiburg, Hugstetterstr. 55, 79106
Freiburg
Tel: 0761-270 3505; FAX: 0761-2703684


From Irene_Tham at hms.harvard.edu Fri Apr 14 15:29:17 2000
From: Irene_Tham at hms.harvard.edu (Irene_Tham)
Date: Fri, 14 Apr 2000 16:29:17 -0400
Subject: putting surface markers in fixed cells
Message-ID: <v04003a00b51d2ebcaeda@[128.103.82.186]>


dear flowers,

once cells are stained with surface markers and fixed, can you put
additional surface markers back into the same sample for detection? i was
told that you can with some Ab. if so, which Ab? Can one reverses the
effect of paraformaldehye? Any suggestions will be appreciated.

irene

Irene C. Tham
Harvard Medical School - NERPRC
Department of Comparative Pathology
1 Pine Hill Drive
Southborough, MA 01722
Office: 508-786-1412
Lab: 508-624-8051
Fax: 508-624-8181




From StevenMerlin at compuserve.com Fri Apr 14 13:24:23 2000
From: StevenMerlin at compuserve.com (Steven Z. Merlin)
Date: Fri, 14 Apr 2000 14:24:23 -0400
Subject: Companies that Lease Lab Equipment
Message-ID: <200004141424_MC2-A13F-1A97@compuserve.com>


On 14 April Phil Barren asked:

>>Does anyone happen to know of a company that leases Lab equipment??

Comdisco leases lab equipment of all types including flow cytometers for
both short and long term leasing. They also sell refurbished lab
instruments and cytometers and have offices worldwide.

Check their web site at: http://www.comdisco.com

or contact them in the USA at 1-800-272-9792

Comdisco Inc
611 North River Road
Rosemont, IL 60018



From tessien at ircm.qc.ca Fri Apr 14 12:20:01 2000
From: tessien at ircm.qc.ca (Nathalie Tessier)
Date: Fri, 14 Apr 2000 13:20:01 -0400
Subject: murine megakaryocytes staining
Message-ID: <38F75341.E4DAEE2B@IRCM.qc.CA>




--
Greetings everyone,

We have to sort murine megakaryocytes. Can anyone recommend a good
staining and is
Hoescht 33342 staining a good solution to identify that population of
less than 1%. In other words,
can we really discriminate that 6N and more population ???? Would it be
better to enrich the bone marrow by depleting other population by MACS.

Thanks for your HELP


Nathalie Tessier, M.Sc.
Head of Flow cytometry Service
Institut de recherches cliniques de Montr?al
110 west, Pine Avenue
Montreal, Quebec, Canada
H2W 1R7
tel: 514-987-5608
fax:514-987-5736




From rhester at jaguar1.usouthal.edu Fri Apr 14 11:05:19 2000
From: rhester at jaguar1.usouthal.edu (ray hester)
Date: Fri, 14 Apr 2000 11:05:19 -0500
Subject: activated rat BAL macrophages, pt. 2
Message-ID: <LPBBKDFDLBFPLGEIAJEGKEHNCAAA.rhester@jaguar1.usouthal.edu>




Hi,

Somone suggested that to detect activated macrophages in rat bronchoalveolar
lavage we could use:

1. anti rat mononuclear phagocyte antibody, 1C7 [with a cellular
distribution said to be similar to CD68 (ED1 antigen)]

plus, either,

2. an anti Ia antibody such as RT1B,

or,

3. anti rat OX-40L (described as a member of the TNF ligand superfamily)

Cost-wise and time-wise it would be best, of course, if we could use two
antibodies instead of three and I wondered if anyone agrees with this
conclusion, and, if so, would you pick RT1B or anti OX-40L to go along with
1C7?

Thanks in advance.

Ray Hester
Univ. of South Alabama



From oamfilho at cpqrr.fiocruz.br Mon Apr 17 08:24:10 2000
From: oamfilho at cpqrr.fiocruz.br (Olindo Assis)
Date: Mon, 17 Apr 2000 10:24:10 -0300
Subject: unexpected T and B phenotypes after culture
References: <A77ECB5409F7D111903E0000F86007CC0474CA78@exchange5.utmb.edu>
Message-ID: <001901bfa870$3975fae0$380983c8@cpqrr.fiocruz.br>


Ol? Hai Qi,
Thank you very much for your reply. I agree with you that B220 is not an
absolute marker for B cells. However, what is so intriguing for me is why in
my stimulated cultures, where I have an intense proliferation and a larger
amount of "blast-large"cells, I do not see this phenomena. Then, if
CD4+B220+ cells are activated T cells why in my Ag-stimulated cultures where
I see proliferation (by thimidin incorporation assay) I do not see this
unexpected phenotype.
Many Thanks (muito obrigado),

Olindo





From dustin at pathbox.wustl.edu Fri Apr 14 18:23:21 2000
From: dustin at pathbox.wustl.edu (Michael Dustin)
Date: Fri, 14 Apr 2000 18:23:21 -0500
Subject: analysis of overlap/segregation betwee two colors in confocal image data
Message-ID: <200004142320.SAA07662@pathbox.wustl.edu>


We are doing multiple color imaging of molecules involved in T cell
activation. We need to develop or employ an existing index for the degree
of overlap (or segregation) between two different molecules that are labeled
with different dyes and imaged with a confocal microscope. One idea would
be to start with a scatter plot (FL1 vs FL2) of the type used for two color
flow
cytometry where individual pixels would be plotted to define regions with
high overlap (on diagonal lower left to upper right) or areas with low
overlap (off diagonal). We can generate lists of pixel values, but need to
translate the text files to a form readable by flow cytometry software like
Cell-quest to get the plots. Is there software to go from Excel files to
the FCS format? Has this problem been dealt with differently by others?
Thanks for any advice.

Michael L. Dustin, Ph.D.
Associate Professor of Pathology
Washington University School of Medicine
660 S. Euclid Ave
Campus Box 8118
St. Louis, MO 63110
Office- (314) 362-9618
Lab- (314) 362-8719
Fax- (314) 362-8888
Jerri Smith, Admin Assist. (314) 362-8740



From BSNEWSOM at txccc.org Fri Apr 14 13:39:26 2000
From: BSNEWSOM at txccc.org (Newsom, Brian S.)
Date: Fri, 14 Apr 2000 13:39:26 -0500
Subject: Cell surface staining and ethanol fixation
Message-ID: <8166BB0493BCD211B1540008C75690E3BAB9FC@GIBSON>


I have had only limited work in this but usually EtOH fixation after a
congugated primary stain does not work very well. What I used to do is stain
cells, fix in 1% paraformaldehyde, wash once and then fix in EtOH, that
seemed to preserve the AB stain much better while still giving good CVs for
the DNA. I have never tried storing the cells after EtOH fixation.

Brian Newsom
Technical Application Specialist
BD/Phamingen

-----Original Message-----
From: oes at rsp.is
To: cyto-inbox
Sent: 4/13/00 5:42 AM
Subject: Cell surface staining and ethanol fixation


hi there

I would like to know wether it is is ok to stain cells for cell surface
markers fix them with ethanol keep them in a freezer for one month and
then stain for DNA content using PI. What effect would that have on my
cell surface markers in the Flowcytometry to fix the cells in ethanol

Hope some one will respond a.s.a.p.

yours

?lafur E Sigurj?nsson
oes at rsp.is



From hms at shapirolab.com Fri Apr 14 17:13:53 2000
From: hms at shapirolab.com (Howard Shapiro)
Date: Fri, 14 Apr 2000 18:13:53 -0400
Subject: UV (280 nm) excitation in flow for lanthanides?
In-Reply-To: <v03020900b51bb8d0fc51@[18.63.2.76]>
Message-ID: <4.2.0.58.20000414180833.00957e90@shell1.shore.net>


Dane Wittrup writes:


>Does anybody have their flow instrument set up for excitation from 280-300
>nm? We'd like to excite a lanthanide bound in calmodulin, using Trp as the
>donor to the ion.

There aren't a lot of people who have lasers that will do those deep UV
lines - the only publication in this area which comes to mind was from the
Jovins in Goettingen in the late 1970's, looking at Trp fluorescence in
proteins.

However, if you're really interested and have the laser (which would
presumably be on an optical bench) and optical filters, I could bring over
the rest of a cytometer. You might also be able to scrounge the
do-it-yourself cytometer parts from Penny Chisholm's lab, which is even
closer to you.

Of course, the real problem might be the lanthanide lifetime; as I recall,
this runs into many microseconds, while the typical dwell time in the beam
of a cytometer is only a few microseconds. Call if you're interested
(617-783-8392 or 617-965-6044).

-Howard





From BRHall at compuserve.com Fri Apr 14 12:02:38 2000
From: BRHall at compuserve.com (Brian Hall)
Date: Fri, 14 Apr 2000 13:02:38 -0400
Subject: Sales position, UK
Message-ID: <200004141303_MC2-A13B-5C8C@compuserve.com>


Cytomation GmbH, the European subsidiary of Cytomation Inc, has the
following instrument sales position open in the UK.

Cytomation GmbH sells and supports the MoFlo high-performance cell sorter
throughout Europe, with its headquarters in Freiburg, Germany. We are now
seeking a full-time salesperson to represent the MoFlo instrument and other
Cytomation products in the UK and Ireland. In keeping with MoFlo's
established position as the premier cell sorter on the market, we need an
individual with outstanding technical knowledge, a passion for the
highest-quality instrumentation, and - most important - a high level of
integrity. We would prefer someone located in or near London, with at least
a few years experience in the flow cytometry business, and with a strong
scientific background. The pay package will be attractive and the
environment of a small company setting the standard that the others follow
has its own rewards. If you are - or if you know someone who may be -
interested in this position, please contact us via your preferred medium.
To find out more about Cytomation, take a look at www.cytomation.com.

Regards,

Brian Hall
Cytomation GmbH
+49-761-559-7370
+49-761-559-7375 fax
+49-171-581-3377 mobile


From Marta.Borg at astrazeneca.com Mon Apr 17 08:58:22 2000
From: Marta.Borg at astrazeneca.com (Marta.Borg@astrazeneca.com)
Date: Mon, 17 Apr 2000 15:58:22 +0200
Subject: EXPOv2 - EXPO32
Message-ID: <052EBD86E10ED41187870001FA7E1D0505B5DD@se-drc-mail2.draco.se.astra.com>


Hi Grant and other EXPO users,

As you brought up the issue, I can tell, I know about the downloading
possibility for EXPO2 and I have done a try, actually for no use. As soon as
I slightly modified and saved a modified protocol, the EXPOv2(Build 320)
crashed, and I had to reinstall the old version EXPOv2(Build 300, not
crashing on the instrument but during analysis for the same reason). Beckman
told me that they have a new version - EXPOv32- available if I will by it.
My question is, how can I know that EXPOv32 is better than EXPOv2 and why
should I pay once more when I newer got what I paid for from the beginning,
an adequate software? (And I made the mistake to by 12 copies!)
Is there anyone out there who has the same problem?

Please all EXPO users, speak up!

Tanks
Marta
____________________________________________
AstraZeneca R&D Lund
Biosciences, S-22187 Lund, Sweden
Tel: +46 46 33 66 18 Fax: +46 46 33 69 70
E-mail: Marta.Borg at astrazeneca.com

-----Original Message-----
From: Grant.Howes at coulter.com [mailto:Grant.Howes at coulter.com]
Sent: onsdag, april 12, 2000 19:50
To: cyto-inbox
Subject: RE: Cytometry Companies and the Internet



As I work for one of the Cytometry Companies, I would not normally respond
to
the bulletin board. However, as you raise the issue of software upgrades, I
thought I should mention that one of our software packages, EXPO2 software
(build 320), is available for download from the following address:

www.appliedcytometry.com


With best regards,

Grant Howes
Marketing Manager, Cytometry Instrument Systems,
Beckman Coulter, Miami



From hodgeg at mail.wch.sa.gov.au Sun Apr 16 17:35:19 2000
From: hodgeg at mail.wch.sa.gov.au (Hodge, Greg (HAEM))
Date: Mon, 17 Apr 2000 08:05:19 +0930
Subject: annexin V dim cells
Message-ID: <200004162237.IAA13347@adl0133.systems.sa.gov.au>




> ----------
> From: shawn_jackson
> Sent: Wednesday, 12 April 2000 23:49
> To: Cytometry Mailing List
> Subject: annexin V dim cells
>
>
> hey flow-ers
>
> I have a question concerning the occurrence of annexin V-dim cells vs.
> bright
> cells. I need help with references and/or data experience!
>
> I am staining peripheral blood from mice infected with vaccinia, and then
> staining the CD8 cells with annexin V-fitc. I see a population of annexin+
> cells extending even up past the 2nd log at many time points, but at
> certain
> times I see very bright cells up at the 3rd log.
>
> I have been warned that only these very bright cells are truly AV+, cells
> with
> any intentions whatsoever of doing anything related to a death process,
> while
> the dimmer cells represent actively proliferating cells only, with no
> intentions of dying. and these dimmer cells are likely to return to an AV-
> population quickly.
>
> Could anyone help me with references or personal experince addressing the
> status of these AV dim cells in contrast to bright ones?
>
> thanks!
>
> shawn jackson
> grad student
> harvard univ
> boston, ma
>
> Hello Shawn,
> I have recently described early apoptotic neutrophils from peripheral
> blood as Ann V dim and late apoptotic neutrophils as Ann V bright (Optimal
> storage conditions for preserving granulocyte viability as monitored by
> Annexin V binding in whole blood. J Immunol Methods 225 (1999) 27-38.
> G.Hodge et al). You could try similar techniques with 7-AAD to determine
> if the CD8 Annex V dim cells are early apoptotic cells.
> Regards Greg Hodge. (hodgeg at mail.wch.sa.go.au).
>


From pxpetit at cochin.inserm.fr Sat Apr 15 06:26:36 2000
From: pxpetit at cochin.inserm.fr (Patricex Petit)
Date: Sat, 15 Apr 2000 13:26:36 +0200
Subject: Superoxid measurements
Message-ID: <l03130301b51df9cd18cb@[193.52.75.146]>


Dear Virginia,

Providing you want measure H2O2, DCFH-DA is very usefull if you have the
proper controls....
And HE will be nice for superoxide anions measurements.

Usefull references

1. Rothe, G., and G. Valet. 1990. Flow cytometric analysis of respiratory
burst activity in phagocytes with hydroethidine and
2',7'-dichlorofluorescin. J. Leukocyte Biol. 47: 440-448.

2. Zamzami, N., P. Marchetti, M. Castedo, C. Zanin, J.-L. Vayssi?re, P.X.
Petit, and G. Kroemer. 1995. Reduction in mitochondrial potential
constitutes an early irreversible step of programmed lymphocyte death in
vivo. J. Exp. Med. 181: 1661-1672.

3. Zamzami, N., P. Marchetti, M. Castedo, D. Decaudin, A. Macho, T. Hirsch,
S.A. Susin, P.X. Petit, B. Mignotte, and G. Kroemer. 1995. Sequential
reduction of mitochondrial transmembrane potential and generation of
reactive oxygen species in early programmed cell death. J. Exp. Med. 182:
367-377.

4. Budd, S.L., R.F. Castilho, and D.G. Nicholls. 1997. Mitochondrial
membrane potential and hydroethidine-monitored superoxide generation in
cultured cerebellar granule cells. FEBS Lett 415: 21-4.

Good luck

Patrice



The message was
Thanks very much to those who sent the extremely useful replies
to my request for elastase activity in neutrophils. Now I am
wondering if anyone knows of any flow methods to measure
superoxide and myeloperoxidase activity in neutrophils.
Any comments are appreciated.

Virginia

?????????????????? \ | /
?????????????????? (o o) \ | /
_______________oOo_____oOo_____oOo_? (o o)__oOo________________

Dr. Petit Patrice X.
Institut Cochin de G?n?tique Mol?culaire
INSERM U129 - CHU Cochin Port-Royal
24, rue du Faubourg Saint-Jacques
F-75014 Paris, France.

Tel: 33 01 44 41 24 11
Fax: 33 01 44 41 24 21
E-mail: pxpetit at icgm.cochin.inserm.fr




From thomas at tritechinc.com Fri Apr 14 10:29:28 2000
From: thomas at tritechinc.com (Joanne Thomas)
Date: Fri, 14 Apr 2000 11:29:28 -0400
Subject: CD4/CD8 coexpression
Message-ID: <003301bfa626$397fad20$1801a8c0@jill.tritechinc.com>

Hi All!
It seems my statement of being able to wash off this coexpression has caused a flurry of requests for more information and questions about the phenomena. We were clued in after reading the following reference: "Artifactual Staining of Monoclonal Antibodies in Two Color Combinations is due to an Immunoglobulin in the Serum and Plasma" Nicholson, JK et. al.; Cytometry 1994 Sept 15;18(3):140-146. Check it out.

Joanne Thomas, M.S.
Director of Operations
TRITECH Field Engineering
2014 Renard Court, Suite I
Annapolis, MD 21401
1-800-886-7004 (USA)
1-410-266-1522
410-266-0935 (FAX)
thomas at tritechinc.com
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From Grant.Howes at coulter.com Fri Apr 14 15:08:51 2000
From: Grant.Howes at coulter.com (Grant.Howes@coulter.com)
Date: Fri, 14 Apr 2000 16:08:51 -0400
Subject: CellProbe Reagents
Message-ID: <852568C1.006E69AB.00@ctcffan6.coulter.com>


Thank you for your interest in Beckman Coulter CellProbe Reagents. You are
correct in your observation that 26 of the original 38 CellProbe reagents have
been discontinued. However, 12 CellProbe reagents remain for sale and are
highlighted in our 2000 Cell Analysis Catalogue. As with every product
discontinuation, a retrospective study is conducted and our results indicated
that without specific applications, the market for certain CellProbes was not as
large as initially thought. It was not due to the acquisition of Coulter
Corporation by Beckman Instruments.

Mara Neal Ends
Marketing Manager
Cytometry Reagents
Beckman Coulter, Inc.
===============================================================================
Christina,

Upon first attempt the link you provided did not work for me. I origionally
was going to respond to Virginia with information about CellProbe; but, when
I looked the other day, I found a list of about ~25-30 of Coulter's
CellProbe reagents on a DISCONTINUED PRODUCTS list.

Could a rep from Beckman/Coulter please respond to this list with an
explaination regarding why these reagents are being discontinued? Are all
of the CellProbe reagents discontinued? Is it because of lack of sales?
Does it have something to do with the Beckman takeover? I understand the
reagents have been critisized because they are susceptable to non-specific
protease (?aminopeptidase) attack. Can anyone comment about their
experience with the non-specific protease lability? Does that have anything
to do with their discontinuation?

Curious in flowland...

Ty Lee
-----Original Message-----
From: Christina McCowan <c.mccowan at pgrad.unimelb.edu.au>
To: cyto-inbox
Date: Friday, April 07, 2000 2:19 PM
Subject: Re: elastase in neurtophils


>
>Virginia,
>
>You might like also to check out the Cell Probe reagents marketed by
>Beckman Coulter.
>
>http://134.217.3.35/coulter/cytometry/CellProbe-Reagents/cp-index.asp
>
>
>Christina McCowan
>
>
>At 18:49 05-04-2000 -0500, you wrote:
>>
>>Virginia,
>>
>>I have not tried this; but, you micht consider looking at this
possibility.
>>http://www.phiphilux.com/elas1.htm
>>I think this is suppose to be a cell permeable substrate that changes
>>fluorescent properties upon protease cleavage.
>>Ty Lee
>>
>>
>>-----Original Message-----
>>From: Virginia M Litwin <virginia.litwin at bms.com>
>>To: cyto-inbox
>>Date: Wednesday, April 05, 2000 5:12 PM
>>Subject: elastase in neurtophils
>>
>>
>>>Does anyone know of a method to measure elastase in neutorphils?
>>>
>>
>




From hms at shapirolab.com Sat Apr 15 19:32:10 2000
From: hms at shapirolab.com (Howard Shapiro)
Date: Sat, 15 Apr 2000 20:32:10 -0400
Subject: PI deactivation
In-Reply-To: <B51BAD9F.B59%kbahjat@ufl.edu>
References: <38F4E2B6.86646AB9@zoo.uvm.edu>
Message-ID: <4.2.0.58.20000415201550.009572d0@shell1.shore.net>


Keith Bahjat writes (re propidium toxicity)-

>How dangerous is a membrane impermeable dye?? Unless it can resurrect dead
>epithelial cells, it should not have access to the DNA of living cells, and
>thus shouldn't be much of a safety hazard.
>
>We always wipe everything down with ethanol, but I've yet to see data
>showing membrane impermeable DNA intercalating dyes cause problems for
>living cells. I think the MSDS claims are based on a general fear of
>anything that has the word "DNA" in it.
>
>Anyone have data to contradict this??

The funny thing is that, according to data on Molecular Probes' safety
sheets, the LD50 in mice for the propidium, which is supposedly
membrane-impermeant, is lower than that for ethidium, which is
membrane-permeant (ethidium is usually pumped out of viable cells, which is
why it often seems to work in dye-exclusion viability tests). So, either
propidium isn't membrane-impermeant all the time, or it may be metabolized
in vivo to something which is.

Note that the terms I use here with respect to the dyes are permeant and
impermeant, describing things which get through and don't, as opposed to
permeable and impermeable, describing membranes which do and don't let
things get through. Membranes are normally impermeable to propidium, which
is impermeant to intact membranes.

The other ringer in this game is that there are circumstances under which
cells become transiently permeable to propidium and structurally similar
dyes (e.g., TO-PRO-1 and TO-PRO-3) without loss of viability. This happens
during scrape loading of cells with various normally impermeant compounds,
during electroporation and other treatments used in transfection, and, in
at least some bacteria, after exposure to sublethal concentrations of some
antibiotics (Novo et al, Antimicrobial Agents and Chemotherapy 44:827-34,
2000). The bacterial response is interesting because it may provide a back
door through which compounds with minimal host toxicity may be introduced
to kill otherwise antibiotic-resistant organisms. If host cell
permeability is variable in the normal scheme of things, this therapeutic
approach becomes harder to implement, but we're still looking into it.

All that said, while I wouldn't substitute propidium for paprika, I
wouldn't get paranoid about it, either.

-Howard




From mgumanovskaya at PharMingen.com Fri Apr 14 15:14:47 2000
From: mgumanovskaya at PharMingen.com (mgumanovskaya@PharMingen.com)
Date: Fri, 14 Apr 2000 13:14:47 -0700
Subject: annexin V stain questions
Message-ID: <852568C1.006F44B6.00@FLKSSMTP01.fl.bdx.com>





Dear Eric and all colleagues,


Thank you for your replays to this question. I am pleased to hear that your
response corroborates what we at PharMingen Technical Service explain to our
customers regarding the issue of washing away free Annexin before analysis. We,
and others, have found it unecessary to do a wash step because unbound Annexin V
falls outside the scatter profile of cells.

Thank you very much to everybody who responded. We sometimes refer our customers
to the expertise of the members of this board if more information is needed.

Best Regards,

Dr. Marina Gumanovskaya
Technical Service Scientist
BD PharMingen






Eric Miller <millere at icrf.icnet.uk> on 04/12/2000 12:49:08 AM

To: cyto-inbox
cc: (bcc: PMGTechServices/SDCA/BDX)
Subject: Re: annexin V stain questions





There is one very good reason why the unbound stain causes no problems:
threshold. If you are thresholding on FSC then it is highly unlikely that
any unbound dye molecules will be large enough to trigger a pulse. Not
washing out unbound stain does feature in many protocols, such as
Vindelov's DNA protocol.

Eric P Miller
Edinburgh Medical Oncology Unit

"Everyone can be correct, all at the same time. That's the
thing about quantum."
Terry Pratchett, Lords and ladies

On Mon, 10 Apr 2000, Jamie Brewer wrote:

>
> I am writing to inquire about an annexin V stain I am used today from
Pharmingen. After
> calling the techinical support service as Pharmingen, I was given this E-mail
> address to correspond to with my unanswered question. The confusion comes in
that
> the Pharmingen protocol does not have me wash off any unbound annexin prior to
flow
> cytometry analysis. This is completely contradictory to any fluorescent stain
I
have ever
> used. Typically, the unbound stain fluoresces when analyzed, losing any
specificity the
> system had. My results seemed to turn out as expected today but I don't
understand why
> the unbound stain is not causing me a problem. I have been unable to find any
reference
> to the annexin being "active" only in the bound form as opposed to the unbound
form
> or any statement indicating the something in the binding buffer inactivates
unbound
> annexin. The technician at Pharmingen didn't have any idea why the unbound
stain is
> not causing a problem. Do you have any understanding of this? Also, the
protocol does
> not offer any option to fix the cells, something which I really need to do
since I
> am using potentially infectious human cells in a common flow cytometry
facility. The
> Pharmingen representative said that she knew of some people who had fixed the
cells
> and it worked alright but not as well as if the cells were not fixed. Do you
have
> any advice/suggestions?
>
> Please correspond to:
> Jamie Brewer
> jbrewer at hsc.wvu.edu
> Microbiology/Immunology Department, Mary Babb Randolph Cancer Center, West
Virginia
> University
>







From haqi at utmb.edu Fri Apr 14 21:03:30 2000
From: haqi at utmb.edu (Qi, Hai)
Date: Fri, 14 Apr 2000 21:03:30 -0500
Subject: unexpected T and B phenotypes after culture
Message-ID: <A77ECB5409F7D111903E0000F86007CC0474CA78@exchange5.utmb.edu>


It is not surprising. I believe you will see double positive cells, and they
are primarily ACTIVATED T cells (this is potentially why you find they are
bigger, assuming not too many doublets are there). Further, you may see T
cells expressing different levels of B220. In my experience, this phenomenon
is also not specific to cells in culture. Overall, I don't think B220 is
really an absolute marker for B cells. Hopefully experts out there will
comment on this.

To gate out cell aggregates, I think you can plot pulse height vs width of
your CD4/CD8 channel (several washes with ice-cold staining buffer should
also help).

Hai Qi
Pathology, UTMB

-----Original Message-----
From: Olindo Assis [mailto:oamfilho at cpqrr.fiocruz.br]
Sent: Wednesday, April 12, 2000 1:01 PM
To: Cytometry Mailing List
Subject: unexpected T and B phenotypes after culture


We have been phenotyping splenocytes from different strains
of mouse before
and after stimulation with bacteria (H. pylori) antigens. We
are basically
labeling the cells using anti-CD4, anti-CD8 and
anti-CD45(B220) PE from
SIGMA. Labeling procedures were done in separated tubes.
Frequently we have noticed that the sum of CD4 + CD45 + CD8
cells from the
unstimulated cultures give us a number over 100%.
It is interesting to observe that this phenomena is observed
only in
unstumulated cells. Cultures in the presence of H. pylori
antigens does not
show this problem. Moreover it was observed for all mouse
strains evaluated
independent of the age. We have used Balb/C, C3H/HeN and
C57/BL6.
It is interesting to observe that these overestimation is
observed only in
the region corresponding to larger cells. We first suggested
that this could
be due to the presence of doublets of CD4 and CD45 cells.
However it could
be also due to the presence of cells co-expressing both
phenotypes.
In order to solve this we are currently evaluating the
presence of double
labeled cells using anti-CD4 FITC and anti-CD45 PE in the
same tube.
However, if we find double labeled cells we still have the
question whether
they are representing doublets or bi-phenotypic cells.
Does any one have experience of phenotypic analysis of T and
B splenocytes
after control cultures? Any suggestions?
Is that possible that CD45 (B220) could be a marker for
cultured CD4 cells?


Any help is appreciated,

Olindo



From BSNEWSOM at txccc.org Fri Apr 14 18:40:12 2000
From: BSNEWSOM at txccc.org (Newsom, Brian S.)
Date: Fri, 14 Apr 2000 18:40:12 -0500
Subject: Sorting CD4+CD3+ cells
Message-ID: <8166BB0493BCD211B1540008C75690E3BABA03@GIBSON>


Joy,

For sorting purposes you will not be able to use the CD3/8/4 set from BD
unless the CD4 is a PerCP-Cy5.5 conjugate, the regular CD4 PerCP does not
show up on the sorter due to the high laser power (unless you are using a
FACSort). The Coulter reagents should work if you have the correct filters.
Some filter sets do not work well in differentiating the PE/ECD/PC5
combination-and the CD45 is probably unneccesary.

Here is what you need to do in a nutshell: gate on a CD3 vs. SSC to get a
popluation of CD3+SSC(low) cells (T-cells), you can also gate on FSC vs SSC
in addition to clean it up a little more. Using that gate look at CD4vsCD8
and you should be able to pick out the populations of choice. Your logical
gating strategy for the sorter will be the same as it is on an analyzer.
Gate on CD3+/SSC(low)/CD4+ cells to sort one way and CD3+/SSC(low)/CD8+
cells to sort the other way. Unless you have a MoFlo 4-way sorter you will
have to do two separate sorts since you want three populations. The third
population will be a gating strategy to isolate CD3+/SSC(low)/CD4-CD8-
cells.

For fluorochromes, I would suggest FITC/PE/PE-CY5 or FITC/PE/PerCP-Cy5.5.

Brian Newsom
Technical Application Specialist
BD/Pharmingen

-----Original Message-----
From: Joy Mundy
To: cyto-inbox
Sent: 4/13/00 7:45 PM
Subject: Sorting CD4+CD3+ cells


Hi everybody,

I am passing the following message on behalf of a researcher in our HIV
research labs. In our lab we are familiar with the analysis of T cell
subsets, currently using the Coulter tetraCHROME reagent. However this
researcher is interested in cell sorting and how to set up the BD flow
sorter to get the cells of interest. As a consequence I am of no use
whatsoever. The antibodies that we currently have at our disposal are
the
Coulter tetraCHROME CD45 FITC/CD4 PE/CD8 ECD/CD3 PC5 and BD CD3/CD8/CD4
together with the BD Tritest control. Analysing the cells are no problem
it's just how th set up the sorter to get the correct cells out!

This is the problem;

Basically what I want to do is as follows
Take 40mls patient blood, ficoll separation.
Stain PBMCs in such a way that I can put them thru a cell sorter and
collect as many cells as possible as need to extract DNA from the
following , to then do a series of PCR reactions to determine integrated
HIV DNA.
Interested in CD4+ lymphocytes (CD3+/CD4+), CD8+ and the small number of
double negative cells (cd4-/CD8-) as these are possibly CD4+ cells that
have down regulated their receptor post HIV infection. I guess the ideal
scenario is to pass the cells thru and collect all CD3+ cells and then
the rest. Then pass thru these CD3+ cells and differentiate between the
4s and the 8s????

Thanks any suggestions welcome !

Joy Mundy
Division of Human Immunology
I.M.V.S.
PO Box 14
Rundle Mall PO
Adelaide 5000
Australia

joy.mundy at imvs.sa.gov.au

Phone (08) 8222-3476
Fax (08) 8232-4092


From oamfilho at cpqrr.fiocruz.br Mon Apr 17 14:04:32 2000
From: oamfilho at cpqrr.fiocruz.br (Olindo Assis)
Date: Mon, 17 Apr 2000 16:04:32 -0300
Subject: putting surface markers in fixed cells
References: <v04003a00b51d2ebcaeda@[128.103.82.186]>
Message-ID: <001201bfa89f$c4422980$380983c8@cpqrr.fiocruz.br>


Dear Irene,

I have used for human cells, anti-CD4 and anti-CD8 FITC labeled (from BD,
Immunotech, Dako, Diatec and Pahrmingen) without any problem to detect cell
surface marker after fixation. However, I know that anti-CD14, anti-CD19 and
anti-CD16 does not recognize fixed cells.

Best regards,

Olindo



From hapsfund at geo.net.ge Mon Apr 17 13:08:49 2000
From: hapsfund at geo.net.ge (HIV/AIDS Patients Support Foundation - Georgia)
Date: Mon, 17 Apr 2000 22:08:49 +0400
Subject: A letter from AIDS Center
Message-ID: <38FB5331.8D35CB83@geo.net.ge>


Dear Messrs,
Would you be please be so kind to briefly answer our question or advise
us who do we have to apply to with the question.
What is and how much is the prognostic importance of CD38 T cells
subsets as activation markers in HIV infection.
Thank you in advance,
Sincerely yours,
David Arveladze




From Gillis_Otten at cc.chiron.com Mon Apr 17 16:58:14 2000
From: Gillis_Otten at cc.chiron.com (Gillis Otten)
Date: Mon, 17 Apr 2000 14:58:14 -0700
Subject: CCR7
Message-ID: <0000AB38.C21364@cc.chiron.com>


This question came up last year, but I could not find answers in the
email archives. Does anyone know of available antibodies to mouse or
human CCR7?


Gib Otten
Chiron Corp
Emeryville, CA


From oamfilho at cpqrr.fiocruz.br Mon Apr 17 12:06:40 2000
From: oamfilho at cpqrr.fiocruz.br (Olindo Assis)
Date: Mon, 17 Apr 2000 14:06:40 -0300
Subject: CD45 x Forward scatter for bone marrow
References: <A77ECB5409F7D111903E0000F86007CC0474CA78@exchange5.utmb.edu>
Message-ID: <000801bfa88f$4d16e900$380983c8@cpqrr.fiocruz.br>


Hi Flowers,
Does anyone know a good reference for CD45 versus side scatter analysis to
differentiate cell populations in bone marrow specimens? I would like to
find a reference with as any details as possible, like dot plots or graphs
showing the discrimination of cell populations. I know I saw something like
this in the past but I do not remember where.
Many thanks in advance,
Olindo




From Zsolt.Juranyi at sanofi-synthelabo.com Tue Apr 18 03:11:40 2000
From: Zsolt.Juranyi at sanofi-synthelabo.com (Zsolt Juranyi)
Date: Tue, 18 Apr 2000 9:11:40 +0100
Subject: rat/guinea-pig neutrophils/eosinophils
Message-ID: <"000418070321Z.WT01083FR/"@MHS>


Does anybody know a flourescent method to stain neutrophils/eosinophils originated
from rat/guinea-pig ? Thanks in advance. Zsolt Juranyi.


From mmorrow at nih.gov Tue Apr 18 10:52:34 2000
From: mmorrow at nih.gov (Matthew Morrow)
Date: Tue, 18 Apr 2000 11:52:34 -0400
Subject: unexpected T and B phenotypes after culture
Message-ID: <v04210100b52232985e68@[165.112.81.130]>


Olindo,

Renno et al (below) describes induced B220 expression on
mouse T-cells stimulated in vivo with SEB. Also, it is known that lpr
and gld mice express CD4-/CD8- T-cells that are B220+. You might be
counting B220 (CD45) on B-cells and T-cells (which you are already
accounting for with CD4 and CD8 therfore counting some of the same
cells twice). It is also expressed on other non-MHC restricted cells
(NK, CTL, and LAK cells).

Renno et al. Eur. J. Immunol. 1998.28:540-547.

Matt
Matthew Morrow, MS, MT (ASCP)
Warren Magnuson Clinical Center
National Institutes of Health
Bethesda, MD
mmorrow at nih.gov/mmorrow at mail.cc.nih.gov
(301) 496-4879


From Joerg.Ueckert at Unilever.com Tue Apr 18 05:00:49 2000
From: Joerg.Ueckert at Unilever.com (Joerg Ueckert)
Date: Tue, 18 Apr 2000 12:00:49 +0200
Subject: analysis of overlap/segregation betwee two colors in confocal image data
Message-ID: <ISSMTP.2000_34_.20000418120049.199B@unilever.com>


Michael,

you might want to try Text2FCS, which converts ASCII data from spreadsheets to
the FCS format. The program was created by Joseph Trotter, works nicely and is
available at Eric Martz' site of Free Flow Software at
http://www.bio.umass.edu/mcbfacs/flowcat.html.

- Joerg


-----Original Message-----
From: Michael Dustin [SMTP:dustin at pathbox.wustl.edu]
Sent: Saturday, April 15, 2000 1:23 AM
To: Cytometry Mailing List
Subject: analysis of overlap/segregation betwee two colors in confocal image
data


We are doing multiple color imaging of molecules involved in T cell
activation. We need to develop or employ an existing index for the degree
of overlap (or segregation) between two different molecules that are labeled
with different dyes and imaged with a confocal microscope. One idea would
be to start with a scatter plot (FL1 vs FL2) of the type used for two color
flow
cytometry where individual pixels would be plotted to define regions with
high overlap (on diagonal lower left to upper right) or areas with low
overlap (off diagonal). We can generate lists of pixel values, but need to
translate the text files to a form readable by flow cytometry software like
Cell-quest to get the plots. Is there software to go from Excel files to
the FCS format? Has this problem been dealt with differently by others?
Thanks for any advice.

Michael L. Dustin, Ph.D.
Associate Professor of Pathology
Washington University School of Medicine
660 S. Euclid Ave
Campus Box 8118
St. Louis, MO 63110
Office- (314) 362-9618
Lab- (314) 362-8719
Fax- (314) 362-8888
Jerri Smith, Admin Assist. (314) 362-8740




From ziltoninca at ieg.com.br Tue Apr 18 07:20:01 2000
From: ziltoninca at ieg.com.br (Zilton Farias Meira de Vasconcelos)
Date: Tue, 18 Apr 2000 09:20:01 -0300
Subject: Parafin cytometry???
Message-ID: <3.0.6.32.20000418092001.00837780@pop3.ieg.com.br>


Hi all,

anyone knows about protocols for parafin lymphom cytometry????

I have some patients in parafin and i don?t know if i could make
immunophenotyping?????

Can anyone help me??? Thanks !!!


Zilton Farias Meira de Vasconcelos
Immunology Laboratory
Bone Marrow Transplantation Unit (CEMO)
Brazilian National Cancer Institute (INCa)
Pra?a Cruz Vermelha,23 - 7o. andar (CEMO) - Centro - Rio de Janeiro - RJ
CEP: 20230-130
e-mail: ziltoninca at ieg.com.br
Phone: 55-021-506-6697
Fax: 55-021-509-2121


From J.H.Schuitemaker at AMC.UVA.NL Tue Apr 18 01:07:09 2000
From: J.H.Schuitemaker at AMC.UVA.NL (Joost Schuitemaker)
Date: Tue, 18 Apr 2000 08:07:09 +0200
Subject: =?iso-8859-1?Q?RE:_TGF=DF_Ab?=
In-Reply-To: <8439A208B822D311B4DD0008C7EAAA0B019BE6FB@phsexch12.partners.org>
Message-ID: <NDBBIMDHELJPNJOENFGPEEEOCCAA.J.H.Schuitemaker@AMC.UvA.NL>



Dear Olson,

IQproducts from Groningen, Holland claims they got a specific TGF? antibody
(human). You can reach them via their website http://www.iqproducts.nl/ or
e-mail general at iqcorporation.nl . Have not (yet) tried it myself.

Best of luck,

Joost Schuitemaker
_________________________________________

J.H.N. Schuitemaker
Research Technician
Dept. Cell Biology & Histology
Cellular Immunology Group
P.O.box 22700
1100 DE Amsterdam
The Netherlands

Tel. +31 (0)20 5664960
Fax. +31 (0)20 8725456

E-mail priv?: J.H.N.Schuitemaker at consunet.nl

-----Original Message-----
From: Olson, Douglas [mailto:DOLSON4 at partners.org]
Sent: vrijdag 14 april 2000 18:33
To: cyto-inbox
Subject: RE:



Does anyone know of an antibody for TGF-beta suitable for flow?

thx,
do

----------------------------------------
Douglas Olson
Experimental Hematology/AIDS Research Center
Massachusetts General Hospital
Harvard Medical School
149 13th Street
Boston, MA 02129
(617)724-2668 - Phone
(617)726-4691 - Fax
Dolson4 at partners.org - Email




From hms at shapirolab.com Mon Apr 17 15:17:36 2000
From: hms at shapirolab.com (Howard Shapiro)
Date: Mon, 17 Apr 2000 16:17:36 -0400
Subject: murine megakaryocytes staining
In-Reply-To: <38F75341.E4DAEE2B@IRCM.qc.CA>
Message-ID: <4.2.0.58.20000417161009.009621c0@shell1.shore.net>


Nathalie Tessier writes-


>We have to sort murine megakaryocytes. Can anyone recommend a good
>staining and is
> Hoescht 33342 staining a good solution to identify that population of
>less than 1%. In other words,
>can we really discriminate that 6N and more population ???? Would it be
>better to enrich the bone marrow by depleting other population by MACS.

In the race to isolate megakaryocytic growth factors, Kuter et al developed
a flow cytometric assay based on 2-parameter flow cytometric measurement of
ploidy and surface antigens using PI and a polyclonal antibody to rat
platelets. They used a Cytomutt, triggering on fluorescence with the
threshold set above 4N, and a rectangular gate to include antibody-positive
events with DNA above 4N. This reliably identified megakaryocytes, even
those cells represented under 1% of the nucleated cells in marrow. I would
imagine you could use the same tricks for sorting, with an anti-platelet
antibody or something more specific and Hoechst 33342 in place of PI - the
only problem would be if the cells pumped out the Hoechst 33342, which
mouse cells may do, but you might be able to block the pump with
trifluoperazine, verapamil, etc.

By the way, they got the growth factor at about the same time as several
larger and better funded groups, thanks in large part to the ploidy assay.

The references are:

Kuter DJ, Greenberg SM, Rosenberg RD: Analysis of megakaryocyte ploidy in
rat bone marrow cultures. Blood 74:1952-1962, 1989
Kuter DJ, Rosenberg RD: Regulation of megakaryocyte ploidy in vivo in the
rat. Blood 75:74-81, 1990
Kuter DJ, Beeler D, Rosenberg RD: The purification of megapoietin: a
physiological regulator of megakaryocyte growth and platelet
production. Proc Natl Acad Sci USA 91:11104-11108, 1994

-Howard






From B.Schmitz at biofrontera.de Tue Apr 18 01:56:50 2000
From: B.Schmitz at biofrontera.de (B.Schmitz@biofrontera.de)
Date: Tue, 18 Apr 2000 08:56:50 +0200
Subject: Sorting of megakaryocytes?
In-Reply-To: <435D520BA7@mm11.ukl.uni-freiburg.de>
Message-ID: <0004180855324300@Mercur.biofrontera.de>


Hallo Dirk,

we established some years ago an easy, reliable and efficient
method for megakaryocyte isolation from human peripheral blood
and bone marrow (percoll gradient centrifugation and subsequent
MACS enrichment and/or FACS). You may contact Dr. Claudia
Wickenhauser for specific applications or the current protocols at
the Institute of Pathology, University of Cologne, Joseph-Stelzmann-
Str. 9, Germany (phone: 49 (0)221-478-5255/6368) or e-mail:
C.Wickenhauser at uni-Koeln.de.
Our procedures are also described in Schmitz et al. Eur J Haematol
1994, 53: 267-275. Moreover, Miltenyi Biotec offers additionally
CD61 MicroBeads for megakaryocyte isolation.
Good luck,
Beate


From hodgeg at mail.wch.sa.gov.au Mon Apr 17 18:41:27 2000
From: hodgeg at mail.wch.sa.gov.au (Hodge, Greg (HAEM))
Date: Tue, 18 Apr 2000 09:11:27 +0930
Subject: annexin V dim cells
Message-ID: <200004172343.JAA02790@adl0133.systems.sa.gov.au>




> ----------
> From: Hodge, Greg (HAEM)
> Sent: Monday, 17 April 2000 8:05
> To: Cytometry Mailing List
> Subject: RE: annexin V dim cells
>
>
>
>
> > ----------
> > From: shawn_jackson
> > Sent: Wednesday, 12 April 2000 23:49
> > To: Cytometry Mailing List
> > Subject: annexin V dim cells
> >
> >
> > hey flow-ers
> >
> > I have a question concerning the occurrence of annexin V-dim cells vs.
> > bright
> > cells. I need help with references and/or data experience!
> >
> > I am staining peripheral blood from mice infected with vaccinia, and
> then
> > staining the CD8 cells with annexin V-fitc. I see a population of
> annexin+
> > cells extending even up past the 2nd log at many time points, but at
> > certain
> > times I see very bright cells up at the 3rd log.
> >
> > I have been warned that only these very bright cells are truly AV+,
> cells
> > with
> > any intentions whatsoever of doing anything related to a death process,
> > while
> > the dimmer cells represent actively proliferating cells only, with no
> > intentions of dying. and these dimmer cells are likely to return to an
> AV-
> > population quickly.
> >
> > Could anyone help me with references or personal experince addressing
> the
> > status of these AV dim cells in contrast to bright ones?
> >
> > thanks!
> >
> > shawn jackson
> > grad student
> > harvard univ
> > boston, ma
> >
> > Hello Shawn,
> > I have recently described early apoptotic neutrophils from peripheral
> > blood as Ann V dim and late apoptotic neutrophils as Ann V bright
> (Optimal
> > storage conditions for preserving granulocyte viability as monitored by
> > Annexin V binding in whole blood. J Immunol Methods 225 (1999) 27-38.
> > G.Hodge et al). You could try similar techniques with 7-AAD to determine
> > if the CD8 Annex V dim cells are early apoptotic cells.
> > Regards Greg Hodge. (hodgeg at mail.wch.sa.go.au).
> >
>


From rleif at rleif.com Mon Apr 17 17:22:08 2000
From: rleif at rleif.com (Robert C. Leif, Ph.D.)
Date: Mon, 17 Apr 2000 15:22:08 -0700
Subject: UV (280 nm) excitation in flow for lanthanides?
In-Reply-To: <4.2.0.58.20000414180833.00957e90@shell1.shore.net>
Message-ID: <NBBBJNOMKDIAJALCEFIJIENDDJAA.rleif@rleif.com>


From: Bob Leif
To: cyto-inbox

My original suggestion (1) to measure Trp fluorescence in flow was to use a
relatively inexpensive flashlamp. The lamp could be triggered by either low
angle light scatter or, as originally suggested, the DC Coulter Effect. If
the cells are flowed slowly, about 500 microseconds observation period, and
the flash is upstream of the optical detection field, you might obtain a
decent signal. The use of deuterium oxide should also be considered. if you
are successful, please let me know. I have other compounds that could be
used.

(1) R. C. Leif; ?A Proposal for an Automated Multiparameter Analyzer for
Cells (AMAC)?. Automated Cell Identification and Sorting, Edited by G. L.
Wied and G. F. Bahr, Academic Press, New York, pp. 131-159 (1970).

-----Original Message-----
From: Howard Shapiro [mailto:hms at shapirolab.com]
Sent: Friday, April 14, 2000 3:14 PM
To: cyto-inbox
Subject: Re: UV (280 nm) excitation in flow for lanthanides?



Dane Wittrup writes:


>Does anybody have their flow instrument set up for excitation from 280-300
>nm? We'd like to excite a lanthanide bound in calmodulin, using Trp as the
>donor to the ion.

There aren't a lot of people who have lasers that will do those deep UV
lines - the only publication in this area which comes to mind was from the
Jovins in Goettingen in the late 1970's, looking at Trp fluorescence in
proteins.

However, if you're really interested and have the laser (which would
presumably be on an optical bench) and optical filters, I could bring over
the rest of a cytometer. You might also be able to scrounge the
do-it-yourself cytometer parts from Penny Chisholm's lab, which is even
closer to you.

Of course, the real problem might be the lanthanide lifetime; as I recall,
this runs into many microseconds, while the typical dwell time in the beam
of a cytometer is only a few microseconds. Call if you're interested
(617-783-8392 or 617-965-6044).

-Howard





From dcoder at u.washington.edu Mon Apr 17 17:01:57 2000
From: dcoder at u.washington.edu (David Coder)
Date: Mon, 17 Apr 2000 15:01:57 -0700
Subject: analysis of overlap/segregation betwee two colors in confocal image data
References: <200004142320.SAA07662@pathbox.wustl.edu>
Message-ID: <03d701bfa8b8$8e32b580$a3725f80@immunol.washington.edu>


Interestingly, the problem was answered by my next email (see below), but you
still want to know just where various classes of pixels are distributed
spatially--i.e., where the pixels are associated with cells and their particular
subcellular structures, or where within a cell are there regions of antigen
expression or coexpression.

Dave
dcoder at u.washington.edu

Date: Sun, 16 Apr 2000 22:56:35 +0100
From: Richard Herd <rahe at unixa.nerc-keyworth.ac.uk>
To: cyto-inbox
Subject: Re: ratio - 2 colour
Message-Id: <l03130300b51fe62c94b4@[209.88.233.113]>
Content-Type: text/plain; charset="us-ascii"

>> In addition, I'm
>>interested in plotting the intensities of a given region of pixels, to
>>compare the Red and green channels pixel by pixel (ie creating a
>>'colocalization graph' if plotted on the x and y axes). Im interested in
>>doing this either on binary (thresholded) images, or on 256 grey scale.


I did this a while ago and it was available on the ftp site
[http://rsb.info.nih.gov/nih-image/]. I don't think
it's still there, but I can prepare you a version of NIH-Image which will
let you plot the XY scatter of pixel intensities for a pair of images in a
stack. Alternatively, the source code if you want to put it in your
version.

Regards - Richard



Richard Herd
Montserrat Volcano Observatory
St Johns
Montserrat
West Indies
email : rahe at ua.nkw.ac.uk
phone : (1) 664 491 5647
FAX : (1) 664 491 2324


----- Original Message -----
From: Michael Dustin <dustin at pathbox.wustl.edu>
To: cyto-inbox
Sent: Friday, April 14, 2000 4:23 PM
Subject: analysis of overlap/segregation betwee two colors in confocal image
data



We are doing multiple color imaging of molecules involved in T cell
activation. We need to develop or employ an existing index for the degree
of overlap (or segregation) between two different molecules that are labeled
with different dyes and imaged with a confocal microscope. One idea would
be to start with a scatter plot (FL1 vs FL2) of the type used for two color
flow
cytometry where individual pixels would be plotted to define regions with
high overlap (on diagonal lower left to upper right) or areas with low
overlap (off diagonal). We can generate lists of pixel values, but need to
translate the text files to a form readable by flow cytometry software like
Cell-quest to get the plots. Is there software to go from Excel files to
the FCS format? Has this problem been dealt with differently by others?
Thanks for any advice.

Michael L. Dustin, Ph.D.
Associate Professor of Pathology
Washington University School of Medicine
660 S. Euclid Ave
Campus Box 8118
St. Louis, MO 63110
Office- (314) 362-9618
Lab- (314) 362-8719
Fax- (314) 362-8888
Jerri Smith, Admin Assist. (314) 362-8740




From claudio.vallan at dkf7.unibe.ch Tue Apr 18 09:33:02 2000
From: claudio.vallan at dkf7.unibe.ch (Claudio Vallan)
Date: Tue, 18 Apr 2000 16:33:02 +0200
Subject: Genome Size assessment
Message-ID: <l03110701b52215a3c1e2@[130.92.148.110]>


Dear flowers,

I was asked to determine the genome size of Phytophtora porri, by measuring
the emission of its propidium iodide (PI) stained DNA and comparing it to
the emission of P.Infestans and E.Coli, for which the genome size is known.
My problem now is, that I do not trust the results for following reasons:

- The Phytophtora and the E. Coli were not treated exactly the same way.
Phytophtora was formaldehyde fixed prior to 70% ethanol treatment while
E. Coli was treated with 70% EtOH. only. Both were treated with DNAse-free
RNAse and stained with 20ug/ml PI, though.
Might the difference in treatment lead to wrong results?

- When we measure cell cycle (e.g. in human and mouse cell lines), for
reasons that are beyond my understanding, now and then some samples which
were treated exactly the same way as the other samples will show a shift of
the G0/1 and G2/M peaks compared to the other samples. (The cells are
guaranteed to have the same amount of DNA. And I saw this with different
users and protocols.) The results are interpretable, but we have to change
the location of the markers (which sometimes is quite difficult, if you
have a shift of the G1 peak and you do not see exactly where the G2/M peak
is). This led me to the conclusion that PI staining might not be ideal to
assess the size of the genome, because of inconsistency of staining.

- When measuring the E.Coli-PI emission I had to dilute them (with BD FACS
Flow) because there were too many of them in the sample. The diluted
bacteria showed a shift to the left of almost one log in emission. Now
either the FACScan will overestimate the fluorescence when charged with too
many cells or, more probably, the staining with PI is very sensitive to
dilutions. So the inconsistencies in the cell cycle explained above could
be due to a drop of sheet fluid falling into the sample.

Does anybody have some useful thoughts on the topic? I will be happy to
summarise any answers.

Happy Eastern to everybody

Claudio

===================================================
Claudio Vallan PhD Phone Lab: 031 / 632 88 76
FACS-LAB DKF Phone Office: 031 / 632 99 68
University of Bern E-Mail: vallan at dkf7.unibe.ch
c/o Institute of Pathology
Murtenstrasse 31 Insel hosptial area only:
3010 Bern Beeper: 181 67 59
===================================================




From kbahjat at ufl.edu Mon Apr 17 21:18:35 2000
From: kbahjat at ufl.edu (Keith Bahjat)
Date: Mon, 17 Apr 2000 22:18:35 -0400
Subject: unexpected T and B phenotypes after culture
In-Reply-To: <A77ECB5409F7D111903E0000F86007CC0474CA78@exchange5.utmb.edu>
Message-ID: <B5213E3A.7A9%kbahjat@ufl.edu>


I can say that in our culture systems following activation, we often find
cells of questionable viability which will bind any antibody you throw in
the tube. You need to make sure you have some marker that these cells are
negative for, like CD13, or F4/80. Something you would never expect to find
on a T cell. When you're in the habit of staining with 4 antibodies, and
looking for cells positive for all 4 antibodies, you're just asking for
trouble.
kb

Keith Bahjat
kbahjat at ufl.edu



on 4/14/00 10:03 PM, Qi, Hai at haqi at utmb.edu wrote:

>
> It is not surprising. I believe you will see double positive cells, and they
> are primarily ACTIVATED T cells (this is potentially why you find they are
> bigger, assuming not too many doublets are there). Further, you may see T
> cells expressing different levels of B220. In my experience, this phenomenon
> is also not specific to cells in culture. Overall, I don't think B220 is
> really an absolute marker for B cells. Hopefully experts out there will
> comment on this.
>
> To gate out cell aggregates, I think you can plot pulse height vs width of
> your CD4/CD8 channel (several washes with ice-cold staining buffer should
> also help).
>
> Hai Qi
> Pathology, UTMB
>
> -----Original Message-----
> From: Olindo Assis [mailto:oamfilho at cpqrr.fiocruz.br]
> Sent: Wednesday, April 12, 2000 1:01 PM
> To: Cytometry Mailing List
> Subject: unexpected T and B phenotypes after culture
>
>
> We have been phenotyping splenocytes from different strains
> of mouse before
> and after stimulation with bacteria (H. pylori) antigens. We
> are basically
> labeling the cells using anti-CD4, anti-CD8 and
> anti-CD45(B220) PE from
> SIGMA. Labeling procedures were done in separated tubes.
> Frequently we have noticed that the sum of CD4 + CD45 + CD8
> cells from the
> unstimulated cultures give us a number over 100%.
> It is interesting to observe that this phenomena is observed
> only in
> unstumulated cells. Cultures in the presence of H. pylori
> antigens does not
> show this problem. Moreover it was observed for all mouse
> strains evaluated
> independent of the age. We have used Balb/C, C3H/HeN and
> C57/BL6.
> It is interesting to observe that these overestimation is
> observed only in
> the region corresponding to larger cells. We first suggested
> that this could
> be due to the presence of doublets of CD4 and CD45 cells.
> However it could
> be also due to the presence of cells co-expressing both
> phenotypes.
> In order to solve this we are currently evaluating the
> presence of double
> labeled cells using anti-CD4 FITC and anti-CD45 PE in the
> same tube.
> However, if we find double labeled cells we still have the
> question whether
> they are representing doublets or bi-phenotypic cells.
> Does any one have experience of phenotypic analysis of T and
> B splenocytes
> after control cultures? Any suggestions?
> Is that possible that CD45 (B220) could be a marker for
> cultured CD4 cells?
>
>
> Any help is appreciated,
>
> Olindo
>



From oamfilho at cpqrr.fiocruz.br Wed Apr 19 06:44:34 2000
From: oamfilho at cpqrr.fiocruz.br (Olindo Assis)
Date: Wed, 19 Apr 2000 08:44:34 -0300
Subject: Many Thants for your help
References: <v04210100b52232985e68@[165.112.81.130]>
Message-ID: <001d01bfa9f4$a2ce32c0$380983c8@cpqrr.fiocruz.br>


Dear Flowers,
I am really thankful to everybody who answered my requests on unexpected T
and B phenotypes after culture and CD45 x bone marrow. I really appreciated
it.
Best regards,
Olindo





From bernaa at excite.com Tue Apr 18 16:24:13 2000
From: bernaa at excite.com (Berna Aslan)
Date: Tue, 18 Apr 2000 14:24:13 -0700 (PDT)
Subject: No subject
Message-ID: <7688277.956093054409.JavaMail.imail@doodle.excite.com>


Hii All
I need a procedure for lenfosit sub group analysis in human broncho alveolar
lavage.

Can anybody help me?

Berna





_______________________________________________________
Get 100% FREE Internet Access powered by Excite
Visit http://freelane.excite.com/freeisp



From Michael_Ormerod at compuserve.com Wed Apr 19 04:46:12 2000
From: Michael_Ormerod at compuserve.com (Michael Ormerod)
Date: Wed, 19 Apr 2000 05:46:12 -0400
Subject: p21
Message-ID: <200004190546_MC2-A1C0-BF8D@compuserve.com>


Can someone give me a reference to a paper describing the flow cytometric
measurement of p21 in cells?

I have searched Medline and have been unable to find anything.

Thanks.

Michael Ormerod
34 Wray Park Road
Reigate RH2 ODE
Telephone: +44 (0)1737 241726
PLEASE NOTE NEW FAX & MOBILE NUMBERS
FAX: +44 (0)1737 226736
Mobile telephone: 07802 293242
Web site: http://ourworld.compuserve.com/homepages/Michael_Ormerod


From dpolakoff at pdl.com Tue Apr 18 14:10:41 2000
From: dpolakoff at pdl.com (Dixie Polakoff)
Date: Tue, 18 Apr 2000 12:10:41 -0700
Subject: Receptors
Message-ID: <fc.004c51fa00448c5a004c51fa00448c5a.448c81@pdl.com>


I have an investigator who wants to assess the number of receptors per
cell. Since I have never done this before so any hints, suggestions,
advice are welcome.

Dixie Polakoff
FACS operator
Protein Design Labs
(510) 574-1528



From tian3w at yahoo.com Wed Apr 19 07:10:20 2000
From: tian3w at yahoo.com (tian tian)
Date: Wed, 19 Apr 2000 05:10:20 -0700 (PDT)
Subject: order CFSE
Message-ID: <20000419121020.3814.qmail@web1705.mail.yahoo.com>



Hi flowers!

I am a PHD student in BeiJing Medical University.I'm
looking to do some CFSE cell tracking experiments in
the next monmth or so and was wondering where would be
the best place to purchase some from
(preferably China, but I should be able to find a
local agency ). Your timely assistance will be greatly
appreciated.

--------------------------------------------
Tian Tian
Department of Immunology,
BeiJing Medical University
tian3w at yahoo.com



__________________________________________________
Do You Yahoo!?
Send online invitations with Yahoo! Invites.
http://invites.yahoo.com


From Zsolt.Juranyi at sanofi-synthelabo.com Wed Apr 19 06:15:15 2000
From: Zsolt.Juranyi at sanofi-synthelabo.com (Zsolt Juranyi)
Date: Wed, 19 Apr 2000 12:15:15 +0100
Subject: rat/guinea-pig haematology
Message-ID: <"000419101711Z.WT09867. 19*/PN=Zsolt.Juranyi/O=FRSANOFI/PRMD=SANOFI/ADMD=ATLAS/C=FR/"@MHS>


Dear Colleagues,

I am looking for any kind of literature/book/haematological atlas that shows the
blood cells of rats and guinea-pigs. Furthermore, I need also some literatures on
the percentage of T/B lymphocytes, granulocytes and other remaining WBCs in rat blood
under normal conditions. Thanks in advance. Zsolt Juranyi.


From markgauci at biotechfrontiers.com Wed Apr 19 01:50:32 2000
From: markgauci at biotechfrontiers.com (Mark Gauci)
Date: Wed, 19 Apr 2000 16:50:32 +1000
Subject: FACSCalibur Sorting
Message-ID: <NDBBLIPHALBPHHDAMLGAOENBFEAA.markgauci@biotechfrontiers.com>


Hi All,

we perform a lot of selective enrichment work using our FACSCalibur, where
the instrument is sorting for long periods of time (ca ~1 hour) at about 200
sort events per second.
I was wondering if this amount of sorting work might unduly damage the
sorting mechanism of the Calibur. Does anyone have experience with this?

Regards,

Mark Gauci
www.biotechfrontiers.com



From ALAA.SAAD at astrazeneca.com Wed Apr 19 09:04:03 2000
From: ALAA.SAAD at astrazeneca.com (ALAA.SAAD@astrazeneca.com)
Date: Wed, 19 Apr 2000 16:04:03 +0200
Subject: CD45 x Forward scatter for bone marrow
Message-ID: <E3394745FB75D011A1E000805FEAC76BA324AB@se-sas-mail1.sa.se.astra.com>


Olindo,

References for CD45 vs. SSC analysis of bone marrow speciemns among others
are:
-"Stelzer GT, Shultz K and Loken MR: CD45 gating for routine flow cytometric
analysis of human bone marrow specimens. Ann NY Acad Sci 667:265-279, 1993".
-" Rainer RO, Hodges L and Stelzer GT: CD45 gating correlates with bone
marrow differential. Cytometry 22:139-145, 1995."


Alaa Saad
___________________________________________________________
AstraZeneca R&D S?dert?lje
Safety Assessment, Clinical Pathology, S-151 85 S?dert?lje, SWEDEN
Tel: +46 8 553 258 93 Fax: +46 8 553 288 23
alaa.saad at astrazeneca.com




From waddi002 at tc.umn.edu Tue Apr 18 18:56:57 2000
From: waddi002 at tc.umn.edu (Kevin Waddick)
Date: Tue, 18 Apr 2000 18:56:57 -0500
Subject: Biotin-streptavidin staining
Message-ID: <38FCF61D.9C8BB9EF@garnet.tc.umn.edu>


Is it really necessary to wash cells between incubation with a
biotinylated antibody and adding streptavidin that is linked to a
fluorochrome? That is, doesn't it work as well if the two are added
together, thereby saving a step? I suppose that I could test this
myself, however I would consider limited attempts by me to be anecdotal.
Others out there may have done actual studies of this and might even
know of tricks to make it work -- assuming that it is not as simple as I
described. That's what I'm hoping, anyway!

Kevin G. Waddick, Ph.D.
Parker Hughes Institute
2657 Patton Road
St. Paul, MN 55113



From echeagaray at sri.org Tue Apr 18 15:42:35 2000
From: echeagaray at sri.org (Echeagaray, Patricia L.)
Date: Tue, 18 Apr 2000 16:42:35 -0400
Subject: Spring Flow Cytometry Meeting - May 3rd - Frederick MD
Message-ID: <D000D917E7BFD311927300508B2C5D05090B62@frcserv.serquest.com>


Meeting Announcement:


The Laboratory of Experimental Immunology

of the

Frederick Cancer Research and Development Center

Presents the Spring

FCRDC Flow Cytometry Workshop

Wednesday, May 3, 2000

10:00 AM - 1:00 PM


Building 549 Auditorium, Ft Detrick, Frederick, Maryland



Moderator - Gordon Wiegand, SAIC, FCRDC


10:00 - 11:00 Applications in Cell Biology, Cell Cycle Kinetics,
and Apoptosis- Gordon Wiegand

11:00 - 11:15 Coffee Break / Visit Industry Representatives

11:15 - 12:15 False Interpretation of Flow Cytometric Data
Leo Burke, Beckman-Coulter

12:15 - ?? Lunch (provided by Beckman-Coulter)



For more information contact Gordon Wiegand @
301-846-1549 or gwiegand at mail.ncifcrf.gov





From Marc.Langweiler at Dartmouth.EDU Wed Apr 19 08:56:04 2000
From: Marc.Langweiler at Dartmouth.EDU (Marc Langweiler)
Date: 19 Apr 2000 09:56:04 EDT
Subject: CD4/CD8 thread
Message-ID: <29962484@dasher.Dartmouth.EDU>

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From Robert.Pyle at rmh.edu Tue Apr 18 16:01:45 2000
From: Robert.Pyle at rmh.edu (Robert Pyle)
Date: Tue, 18 Apr 2000 17:01:45 -0400
Subject: Telomerase Assays
Message-ID: <s8fc9514.092@mail3.rmh.edu>


Last year there was a brief discussion on the mail list regarding the application of
flow cytometry to telomerase assays. There was some discussion of performing FISH
and then analyzing on a flow cytometer. Is anyone out there actually performing
such an analysis at this time or have some experience in this that I might be able
to benefit from? We are considering a flow cytometry based assay for telomerase here
and would love to hear what others might be now doing in this area.

Any comments or experiences will be most appreciated.

R. Haywood Pyle
Supervisor, Cell Analysis Laboratory
South Carolina Cancer Center
7 Med Park Dr.
Columbia, SC 29203
Tel: 803-434-4960
Fax: 803-434-4950
Email: robert.pyle at rmh.edu



From Dana_Levasseur at microbio.uab.edu Wed Apr 19 02:14:42 2000
From: Dana_Levasseur at microbio.uab.edu (Dana Levasseur)
Date: Wed, 19 Apr 2000 02:14:42 -0500
Subject: mouse/human hepatocyte specific marker
Message-ID: <BCC4C6C4DF90D311B40A00A0C92C80270D0CFD@micro.microbio.uab.edu>


Hello All,

Maybe this is a question the flow community can tackle? We are looking for
an antibody specific for hepatocytes. Specifically, we are trying to
differentiate between cells of erythroid versus other origins in fetal liver
and were wondering if a "hepatocyte specific marker" existed. TER119 is the
logical option for the erythroid marker. Also, I know the question was
posed to the group a short time ago, but does there exist an erythroid
marker that can differentiate more primitive precursors such as BFU-E,
CFU-E, or normoblasts? Presumably, TER119 stains these cells, but it also
stains retics. and mature erythrocytes.

Many thanks in advance,


Dana Levasseur
University of Alabama at Birmingham
Bevill Biomedical Research Building, Rm. 870
845 19th Street South
Birmingham, AL 35205

phone: (205)-934-1963
e-mail: dnl at uab.edu


From sw11527 at glaxowellcome.com Tue Apr 18 18:50:24 2000
From: sw11527 at glaxowellcome.com (Witherspoon, Sam)
Date: Tue, 18 Apr 2000 19:50:24 -0400
Subject: DAPI and APC on 2nd laser lines
Message-ID: <17774C9FD541D211A95800805FE6AE9B01F0749E@US4N65>


Hi Group
I'm trying to collect DAPI and APC signals on the second (and third)
laser lines of a FACStar Plus in a manner very similar to that described by
Moore et al in:
Cytometry (32) 1: 57-65 , May 1998. The method describes the
collection of DAPI and APC fluorescence from cells excited by collinear
beams of a mixed gas laser for UV (350-356 nm) and a HeNe laser (633 nm).

In this technical note, the filters adjacent to the relevant PMT's are
described (660DF20 for APC and 450DF20 for DAPI), but the splitter between
the two PMT's is not mentioned.
I have looked around and cannot find a reference as to the "proper
thing to do" in this case.

The DAPI signal is very bright... should I reflect it and filter the
APC fluorescence or should I filter the DAPI and reflect the APC?

Is there a general rule that I could apply based on wavelength regardless of
"brightness"?

Thanks in Advance,

I look forward to your input.

Cheers,
Sam
Sam Witherspoon
sw11527 at glaxowellcome.com

Dept. of Receptor Biochemistry Tel. 919-483-3078
Glaxo Wellcome R&D Page 919-857-7768
5 Moore Dr. Fax 919-483-0585
RTP, NC 27709




From joy.mundy at imvs.sa.gov.au Tue Apr 18 23:51:09 2000
From: joy.mundy at imvs.sa.gov.au (Joy Mundy)
Date: Wed, 19 Apr 2000 14:21:09 +0930
Subject: Diagram to display fluorochrom emission overlap
Message-ID: <000701bfa9ba$e10e86a0$f767140a@ihi31382.imvs.sa.gov.au>


HI,

Does anybody have a good diagram they can reference or send as an
attachement that shows the spectral emission overlap for the following
fluorchromes;

FITC, PE, ECD and PCY5


Thanks

Joy Mundy
Division of Human Immunology
I.M.V.S.
PO Box 14
Rundle Mall PO
Adelaide 5000
Australia

joy.mundy at imvs.sa.gov.au

Phone (08) 8222-3476
Fax (08) 8232-4092



From julmoore at calc.vet.uga.edu Wed Apr 19 09:23:41 2000
From: julmoore at calc.vet.uga.edu (Julie Moore)
Date: Wed, 19 Apr 2000 09:23:41 EST
Subject: CCR7
In-Reply-To: <0000AB38.C21364@cc.chiron.com>
Message-ID: <200004191323.IAA32734@flowcyt.cyto.purdue.edu>


See below...

Date: Mon, 17 Apr 2000 14:58:14 -0700
From: Gillis_Otten at cc.chiron.com (Gillis Otten)
Subject: CCR7
To: cyto-inbox


This question came up last year, but I could not find answers in the
email archives. Does anyone know of available antibodies to mouse or
human CCR7?


Gib Otten
Chiron Corp
Emeryville, CA

Gib--

A clone developed by Lijun Wu at Leukosite (now merged with
Millenium) should be available soon (we hope!) through
Pharmingen. They were in the process of setting up the marketing the
last time I checked (in January). If you talk to Pharmingen and get
a positive response, let us all know!!



Julie Moore, PhD
Center for Tropical and Emerging Global Diseases
AND Department of Medical Microbiology and Parasitology
College of Veterinary Medicine
University of Georgia
phone: 706-542-5789
fax: 706-542-0059
email: julmoore at calc.vet.uga.edu


From WEAVER at CDER.FDA.GOV Wed Apr 19 08:35:06 2000
From: WEAVER at CDER.FDA.GOV (James Weaver 301-594-5879 FAX 301-594-3037)
Date: Wed, 19 Apr 2000 09:35:06 -0400 (EDT)
Subject: unexpected T and B phenotypes after culture
In-Reply-To: <000801bfa4a9$0450dda0$380983c8@cpqrr.fiocruz.br>
Message-ID: <E4477IKR22G3X*/R=A1/R=FDACD/U=WEAVER/@MHS>


We have on rare occasions observed a significant population (~30% vs <
0.5% normal) of mouse periperal blood leukocytes that appear to
co-express TcR-Beta and CD-45R (B-220). This is a transient event as the
same mice sampled 3 days later do not show this population. The CD4 and
CD8 percentages in these mice were normal. I have also heard anecdotal
reports of a similar response following in vivo stimulation with Staph
enterotoxin B.

-Jim Weaver


*************************************************
* *
* James L. Weaver Ph.D. *
* Division of Applied Pharmacology Research *
* Office of Testing & Research *
* CDER MOD-1, FDA *
* 8301 Muirkirk Rd, Laurel MD 20708 *
* *
* Phone: 301-594-5879 *
* Fax: 301-594-3037 *
* Email:WEAVER at CDER.FDA.GOV *
* *
*************************************************




From bunny at cotleur.com Tue Apr 18 23:40:57 2000
From: bunny at cotleur.com (bunny)
Date: Wed, 19 Apr 2000 00:40:57 -0400
Subject: quenching? or not?
Message-ID: <38FD38D8.F326296E@cotleur.com>


Fellow Flowers-

We have a very strange problem and I'm at a loss to explain. My
colleague is staining fresh human LWB with CD3-PERCP and CXCR3-PE. When
the CD3-percp control is run on the Facscan, itlooks ok, comps fine &
nothing remarkable is noted. When the CD3+CXCR3 is run, it appears that
the CXCR3-neg CD3's are GONE. [As in quenched?] In fact, this person was
doing a series of CXCR3 Ab dilutions and the more dilute the CXCR3 Ab,
the more pronounced the CD3 quench.
I reran her samples completely uncompensated-(data posted at this link)

http://www.cotleur.com/CotleurMain.data/Components/facsdata.html

and you see that on left is uncomp'd CD3 alone, and the right-
uncomp'd CD3+CXCR3. ObserveThanks the shift after addition of the CXCR3;
so much so that the plot almost appears to actually be compensated.

So here's the wrench:
1) this stain/combo was the exact stain (except the dilutions) that
they ran many times before, never saw this. The CXCR3 dilutions were
done for the first time yesterday, and did not show this CD3 shift.
(CXCR3 Ab was serially diluted in PBS/2%NCS/+azide, 0.02%).

2) With each increasing ab dilution, the effect is pronounced.

3) the Facscan was just serviced today. Calibrite beads look perfectly
normal, the single stain cells look normal also.

4) CD3 paired with CCR2-PE: does NOT see this.

I am convinced it is something in their prep, they are convinced it is
the instrument (only because their experiment "worked" yesterday, and
did not today- post instrumet service).

Has anyone EVER seen anythiung remotely like this?


All comments welcome!

Bunny






*******************************************************
Bunny Cotleur +*+ Bunny Cotleur
Cleveland Clinic Foundation *+* 2001 Lester RD
Neurosciences NC30 +*+ Valley City, OH 44280
9500 Euclid Avenue *+* 330-483-4800
Cleveland, OH 44195 +*+ bunny at cotleur.com
216-444-1164 *+*
cotleua at ccf.org +*+

*******************************************************
When you do something, you should burn yourself completely, like a good
bonfire, leaving no trace of yourself.
(Shunryu Suzuki)


From simmmmer at yahoo.com Tue Apr 18 17:14:26 2000
From: simmmmer at yahoo.com (Maciej Simm)
Date: Tue, 18 Apr 2000 15:14:26 -0700 (PDT)
Subject: macrophage apoptosis and flow
Message-ID: <20000418221426.1357.qmail@web2004.mail.yahoo.com>


Dear all,

One of the people I'm working with right now is studying apoptosis in
periteneal macrophages using a kit with annexin V monoclonal FITC ab
and propidium iodine.

The cells are autofluorescent. The "unstained" tube has signal in up
to 2nd log.

The stained tubes are VERY HIGHLY BRIGH on FL3 (PI) and I tried to
make things "fit" on the dotplot by lowering fl3 voltage by 200 or
so, but I'm still not getting everything (and also some of the dimmer
cells are squooshed on the axis). The FITC signal is of normal
brighness.

Needless to say we're not getting the same results as the kit
suggests we should given the incubation/conditions. He's gonna talk
to the company. I am posting this message with hopes that some of you
may have seen this method or one similar to it and are willing to
comment.

Any suggestions are welcome

thanks a bunch,

Maciej



=====
`---------------------------------------------`
| Maciej S. Simm | 525 E 68th Street |
| Research Technician | Room N-805 |
| Cornell Medical Center | Tel. 212.746.3428 |
`---------------------------------------------`
| www.cd4cd8.com |
`---------------------------------------------`

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From Nigel.Blackhall at nottingham.ac.uk Wed Apr 19 06:06:24 2000
From: Nigel.Blackhall at nottingham.ac.uk (Nigel Blackhall)
Date: Wed, 19 Apr 2000 12:06:24 +0100
Subject: Genome Size assessment
Message-ID: <s8fda157.073@ccw0m.ccc.nottingham.ac.uk>

** Reply Requested When Convenient **

Dear colleagues,

Claudio Vallan raised a query concerning staining of bacteria with PI.

In the plant world, PI is considered the fluorochrome of choice for genome size determination.

However, it is important that all of the samples have exactly the same treatment. Addition of paraformaldehyde may alter the penetration of the dye into interphase nuclei.

Most people try and use PI at a 'saturating' concentration so that small disturbances of the concentration have minimal effect on the amount of dye bound. We use 50 mg/l. If a sample needs diluting, we use staining buffer to maintain the PI concentration.

Regards

Nigel Blackhall
Experimental Officer, Plant Science Division,
School ogf Biological Sciences,
University of Nottingham,
University Park Nottingham NG7 2RD.
Tel 0115 9515151 x 18501
Fax 0115 9513298
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HTML attachment scrubbed and removed

From enockson at musc.edu Wed Apr 19 09:29:40 2000
From: enockson at musc.edu (Candace Enockson)
Date: Wed, 19 Apr 2000 10:29:40 -0400
Subject: unexpected T and B phenotypes after culture
In-Reply-To: <A77ECB5409F7D111903E0000F86007CC0474CA78@exchange5.utmb.edu>
Message-ID: <154727.3165128980@[128.23.89.125]>


I was glad to see your comment on B220. We are using B220 in various
experiments - on IEL, pyer's patches, lung associated lymph nodes, and
spleen, control and infected and we see all sorts of B220+ populations, dim
and bright. I am not very experienced with this marker and need all the
suggestions this site can offer.

Candace Enockson
Medical University of South Carolina

--On Fri, Apr 14, 2000 9:03 PM -0500 "Qi, Hai" <haqi at utmb.edu> wrote:

>
> It is not surprising. I believe you will see double positive cells, and
> they are primarily ACTIVATED T cells (this is potentially why you find
> they are bigger, assuming not too many doublets are there). Further, you
> may see T cells expressing different levels of B220. In my experience,
> this phenomenon is also not specific to cells in culture. Overall, I
> don't think B220 is really an absolute marker for B cells. Hopefully
> experts out there will comment on this.
>
> To gate out cell aggregates, I think you can plot pulse height vs width of
> your CD4/CD8 channel (several washes with ice-cold staining buffer should
> also help).
>
> Hai Qi
> Pathology, UTMB
>
> -----Original Message-----
> From: Olindo Assis [mailto:oamfilho at cpqrr.fiocruz.br]
> Sent: Wednesday, April 12, 2000 1:01 PM
> To: Cytometry Mailing List
> Subject: unexpected T and B phenotypes after culture
>
>
> We have been phenotyping splenocytes from different strains
> of mouse before
> and after stimulation with bacteria (H. pylori) antigens. We
> are basically
> labeling the cells using anti-CD4, anti-CD8 and
> anti-CD45(B220) PE from
> SIGMA. Labeling procedures were done in separated tubes.
> Frequently we have noticed that the sum of CD4 + CD45 + CD8
> cells from the
> unstimulated cultures give us a number over 100%.
> It is interesting to observe that this phenomena is observed
> only in
> unstumulated cells. Cultures in the presence of H. pylori
> antigens does not
> show this problem. Moreover it was observed for all mouse
> strains evaluated
> independent of the age. We have used Balb/C, C3H/HeN and
> C57/BL6.
> It is interesting to observe that these overestimation is
> observed only in
> the region corresponding to larger cells. We first suggested
> that this could
> be due to the presence of doublets of CD4 and CD45 cells.
> However it could
> be also due to the presence of cells co-expressing both
> phenotypes.
> In order to solve this we are currently evaluating the
> presence of double
> labeled cells using anti-CD4 FITC and anti-CD45 PE in the
> same tube.
> However, if we find double labeled cells we still have the
> question whether
> they are representing doublets or bi-phenotypic cells.
> Does any one have experience of phenotypic analysis of T and
> B splenocytes
> after control cultures? Any suggestions?
> Is that possible that CD45 (B220) could be a marker for
> cultured CD4 cells?
>
>
> Any help is appreciated,
>
> Olindo
>






From sw11527 at glaxowellcome.com Wed Apr 19 07:24:32 2000
From: sw11527 at glaxowellcome.com (Witherspoon, Sam)
Date: Wed, 19 Apr 2000 08:24:32 -0400
Subject: DAPI and APC on 2nd laser lines
Message-ID: <17774C9FD541D211A95800805FE6AE9B01F074A1@US4N65>


Please accept my apologies for having APC on the brain
The reference below was published as using Cy5, not APC.
Still, my desire is to use APC in my application if possible.

Thanks,
Sam Witherspoon

> -----Original Message-----
> From: Witherspoon, Sam
> Sent: Tuesday, April 18, 2000 7:50 PM
> To: 'Cytometry Mailing List'
> Subject: DAPI and APC on 2nd laser lines
>
> Hi Group
> I'm trying to collect DAPI and APC signals on the second (and third)
> laser lines of a FACStar Plus in a manner very similar to that described
> by Moore et al in:
> Cytometry (32) 1: 57-65 , May 1998. The method describes the
> collection of DAPI and [Sam] (was Cy5 not APC) APC fluorescence from
> cells excited by collinear beams of a mixed gas laser for UV (350-356 nm)
> and a HeNe laser (633 nm).
>
> In this technical note, the filters adjacent to the relevant PMT's are
> described (660DF20 for [Sam] Cy5, not APC ) and 450DF20 for DAPI), but
> the splitter between the two PMT's is not mentioned.
> I have looked around and cannot find a reference as to the "proper
> thing to do" in this case.
>
> The DAPI signal is very bright... should I reflect it and filter the
> APC fluorescence or should I filter the DAPI and reflect the APC?
>
> Is there a general rule that I could apply based on wavelength regardless
> of "brightness"?
>
> Thanks in Advance,
>
> I look forward to your input.
>
> Cheers,
> Sam
> Sam Witherspoon
> sw11527 at glaxowellcome.com
>
> Dept. of Receptor Biochemistry Tel. 919-483-3078
> Glaxo Wellcome R&D Page 919-857-7768
> 5 Moore Dr. Fax 919-483-0585
> RTP, NC 27709
>



From DARZYNK at nymc.edu Wed Apr 19 11:34:34 2000
From: DARZYNK at nymc.edu (DARZYNKIEWICZ ZBIGNIEW)
Date: Wed, 19 Apr 2000 12:34:34 -0400
Subject: Join other scientists in support of agricultural biotechnology
Message-ID: <8C3E01568EC0D311AF0600508B0CC32B020F19D6@MAIL>


Dear Collegues,
In the public debate about benefits and possible dangers of genetically
altered plants the voice of scientists is rarely heard. Mass media
concentrate on vocal, often missinformed "activists" who are unable to
understand the principles of modern biotechnology and consider the modified
plants equivalent of a "new Frankenstein" generated by scientists to destroy
the world.
While the possible risks associated with genetically modified plants should
be taken seriously and its safety should be scientifically evaluated, it is
also duty of scientists to inform the public, presenting possible risks and
benefits. Toward this end over 2,000 scientists, many renown names included
have signed a document in support of agricultural biotechnology. The unified
voice of scientists presented to the public may calm their anxiety in this
area.
I appeal, therefore, to you to join the list and sign the document.
It is available under www.agbioworld.org
Zbigniew Darzynkiewicz


From thomas at tritechinc.com Wed Apr 19 10:49:02 2000
From: thomas at tritechinc.com (Joanne Thomas)
Date: Wed, 19 Apr 2000 11:49:02 -0400
Subject: CD45 x Forward scatter for bone marrow
Message-ID: <002301bfaa16$c9ab1c60$1801a8c0@jill.tritechinc.com>


Olindo:
For human BM see -
Stelzer, Schults, and Loken: "CD45 gating for routine flow cytometric
analysis of human bone marrow specimens" Ann. NY Acad. Sci. 677:265, 1993
Borowitz, Guenther, schults, Stelzer: "Immunophenotyping of acute leukemia
by flow cytometric analysis; use of CD45 and right angle light scatter to
gate on leukemic blasts in three color analysis" Am J Clin Pathol 100:534,
1993.

Joanne Thomas, M.S.
Director of Operations
TRITECH Field Engineering
2014 Renard Court, Suite I
Annapolis, MD 21401
1-800-886-7004 (USA)
1-410-266-1522
410-266-0935 (FAX)
thomas at tritechinc.com
-----Original Message-----
From: Olindo Assis <oamfilho at cpqrr.fiocruz.br>
To: cyto-inbox
Date: Tuesday, April 18, 2000 6:34 PM
Subject: CD45 x Forward scatter for bone marrow


>
>Hi Flowers,
>Does anyone know a good reference for CD45 versus side scatter analysis to
>differentiate cell populations in bone marrow specimens? I would like to
>find a reference with as any details as possible, like dot plots or graphs
>showing the discrimination of cell populations. I know I saw something like
>this in the past but I do not remember where.
>Many thanks in advance,
>Olindo
>
>



From DARZYNK at nymc.edu Wed Apr 19 15:42:23 2000
From: DARZYNK at nymc.edu (DARZYNKIEWICZ ZBIGNIEW)
Date: Wed, 19 Apr 2000 16:42:23 -0400
Subject: p21
Message-ID: <8C3E01568EC0D311AF0600508B0CC32B020F19D8@MAIL>


Mike Ormerod asked about p21 measurement by flow cytometry.

Can someone give me a reference to a paper describing the flow cytometric
measurement of p21 in cells?

I have searched Medline and have been unable to find anything.

Thanks.

Michael Ormerod


I presume you mean p21WAF1 rather than p21RAS. We have measured p21WAF1 by
laser scanning cytometry using PharMingen antibody (clone 2G12) (Deptala et
al., Int. J. Oncol., 15: 861-871, 1999).
Zbigniew Darzynkiewicz


From leonidv at crccuse.usherb.ca Thu Apr 20 09:19:51 2000
From: leonidv at crccuse.usherb.ca (Leonid Volkov)
Date: Thu, 20 Apr 2000 10:19:51 -0400
Subject: emission overlap
Message-ID: <a0431010ab524af8943d2@[132.210.157.86]>


Dear Joy Mundy,

>the spectral emission overlap for the following
fluorchromes;

http://fluorescence.bio-rad.com/frames6.htm

I hope this information is helpful

Best regards
--
Dr. L. VOLKOV
Ass. en Cytometrie -Microscopie
Serv. d'Immunologie
Fac. de medecine
3001, 12 av.Nord SHERBROOKE (Quebec); CANADA
J1H 5N4
Tel: (819) 346-1110 ext 4867
Fax: (819) 564-5215
Leonid Volkov <leonidv at crccuse.usherb.ca>


From oamfilho at cpqrr.fiocruz.br Wed Apr 19 14:19:35 2000
From: oamfilho at cpqrr.fiocruz.br (Olindo Assis)
Date: Wed, 19 Apr 2000 16:19:35 -0300
Subject: Receptors
References: <fc.004c51fa00448c5a004c51fa00448c5a.448c81@pdl.com>
Message-ID: <000e01bfaa34$33012e80$380983c8@cpqrr.fiocruz.br>


I know that BD has a system called quantiBright (or QuantiBrite) that can be
used for these purpose.
Also STAGO has a similar product designed to analysis of receptors on
platelets.

Good luck,

Olindo



From hms at shapirolab.com Wed Apr 19 22:30:35 2000
From: hms at shapirolab.com (Howard Shapiro)
Date: Wed, 19 Apr 2000 23:30:35 -0400
Subject: Diagram to display fluorochrom emission overlap
In-Reply-To: <000701bfa9ba$e10e86a0$f767140a@ihi31382.imvs.sa.gov.au>
Message-ID: <4.2.0.58.20000419232546.00968850@shell1.shore.net>


Joy Mundy writes-

>Does anybody have a good diagram they can reference or send as an
>attachement that shows the spectral emission overlap for the following
>fluorchromes;
>
>FITC, PE, ECD and PCY5

There is such a diagram, unique, as far as I know, in that it shows
emission from equal concentrations of antibodies, on p. 164 of the 3rd
Edition of Practical Flow Cytometry. If you can't find a copy of the book
nearby, I might be able to send an equivalent diagram (Wiley has the
copyright on the one in the book).

-Howard



From D.R.LLOYD at bham.ac.uk Thu Apr 20 05:44:56 2000
From: D.R.LLOYD at bham.ac.uk (DR LLOYD)
Date: Thu, 20 Apr 2000 11:44:56 +0100
Subject: anti HRP FITC conjugate
Message-ID: <E12iESc-00062e-00@bham.ac.uk>


Dear all

I have a request for help from a colleague who wants to do some
signal amplification.

Does anyone know of a source (preferably UK) of an FITC
conjugated anti Horse Radish Peroxidase antibody?

Any suggestions will be greatly appreciated; please send replies to;
P.HOLMES.20 at bham.ac.uk

Thanks in advance




David Lloyd
School of Chemical Engineering
University of Birmingham
Edgbaston
Birmingham B15 2TT
England

t; +44 (0)121 414 5267
e; D.R.Lloyd at bham.ac.uk


From oberyszyn.2 at osu.edu Wed Apr 19 13:58:50 2000
From: oberyszyn.2 at osu.edu (Andrew Oberyszyn)
Date: Wed, 19 Apr 2000 14:58:50 -0400
Subject: Talks for Annexin V help
Message-ID: <4.2.0.58.20000419145740.009c6e70@pop.service.ohio-state.edu>


Hi Flow-ers,
I want to thank all those who responded to my question about the Annexin V
staining problem I was having. I passed along the info to the user.

Once again thanx!

Andy

(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)
Andy Oberyszyn, M.S.
The Ohio State University
Analytical Cytometry Laboratory
416 Comprehensive Cancer Center
410 West 12th Avenue
Columbus, Ohio 43210
Tel: 614/292-FLOW(3569)
Fax: 614/292-7335
E-Mail: cytometry at osu.edu
Web Page:
<http://cytometry.med.ohio-state.edu>http://cytometry.med.ohio-state.edu
(-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-)

"What if the Hokey Pokey is really what it's all about?!?"


From djyoung at UCSD.Edu Wed Apr 19 11:59:13 2000
From: djyoung at UCSD.Edu (Dennis J. Young)
Date: Wed, 19 Apr 2000 09:59:13 -0700
Subject: putting surface markers in fixed cells
In-Reply-To: <001201bfa89f$c4422980$380983c8@cpqrr.fiocruz.br>
References: <v04003a00b51d2ebcaeda@[128.103.82.186]>
Message-ID: <3.0.1.32.20000419095913.0083ace0@popmail.ucsd.edu>


I found that what fixative makes a difference. (Beckman)Coulter's Permafix
preserved CD34 antigenicity for one Ab, while BD's FACS Lysing Solution
destroyed it. A different Ab didn't work with either fixative.
(N = 1)


At 04:04 PM 4/17/00 -0300, Olindo Assis wrote:
>Dear Irene,
>
>I have used for human cells, anti-CD4 and anti-CD8 FITC labeled (from BD,
>Immunotech, Dako, Diatec and Pahrmingen) without any problem to detect cell
>surface marker after fixation. However, I know that anti-CD14, anti-CD19 and
>anti-CD16 does not recognize fixed cells.
>
>Best regards,
>
>Olindo
>
>
---
Dennis

Dennis J. Young
Mail:<<mailto:djyoung at ucsd.edu>>
WWW:<<http://cancer.ucsd.edu/SResources/flow.htm>>

Flow Cytometry Core Facility
University of California, San Diego
Internal Medicine Group, Bldg #4, Rm 126
9500 Gilman Drive La Jolla CA 92093-0671
Telephone:(858) 822-0407


From simmmmer at yahoo.com Wed Apr 19 16:37:35 2000
From: simmmmer at yahoo.com (Maciej Simm)
Date: Wed, 19 Apr 2000 14:37:35 -0700 (PDT)
Subject: CD11b expression on Lymphocytes
Message-ID: <20000419213735.22592.qmail@web2003.mail.yahoo.com>


Greetings once again,

We have recently received a blood specimen and were asked to stain
for CD18/11b ( I love it how doctors don't explain the condition of
the patient ahead of time, so we know what to look for )

We found, that when compared to our NC's (N=300+ for this "tube" and
growing) this patient had less +/+ lymphoctes (~30% vs. 12%). The
expression of CD18 was not reduced - all of his leukocytes had >97%.

A question came up: what is the significance of CD18+/CD11b-
lymphocytes? what is the significance of a decrease in the +/+
population?

We do not routinely do CD11a or CD11c - are these mutually exclusive?
I.e. it's either a, b or c but never any of them together?

And finally, is the ligand for CD11b soluble and may have interfered
with our staning?

This staining was done in whole blood (which I just found is a
no-no).

I would appreciate if anyone could share any thoughts on this topic.

Maciej

__________________________________________________
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Send online invitations with Yahoo! Invites.
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From JBarry at picr.man.ac.uk Thu Apr 20 05:12:20 2000
From: JBarry at picr.man.ac.uk (Jeff Barry)
Date: Thu, 20 Apr 2000 11:12:20 +0100
Subject: G0 Markers
Message-ID: <c=US%a=_%p=Paterson_Institu%l=LANCELOT-000420101220Z-122@lancelot.picr.man.ac.uk>


Dear Flow Cytometerist,

I would be grateful for your assistance in answering a question that I
have been asked by one of our researchers concerning the
identification of G0 cells. We have been investigating G0 cells based
on Pyronin Y and Hoechst staining and we have been identifying G0
cells as those that have very low Pyronin Y content and that also
appear in the G0/G1 Hoechst peak. What my colleague would like to know
is ....

"How have people confirmed that cells staining with low Pyronin Y and
low Hoechst are G0? Has anyone isolated cells and looked for any other
markers of G0?"

Any comments or references on this matter would be appreciated.

Happy Easter,

Jeff Barry
Paterson Institute for Cancer Research
Manchester
UK



From alexandra.dorn-beineke at ikc.ma.uni-heidelberg.de Thu Apr 20 04:56:34 2000
From: alexandra.dorn-beineke at ikc.ma.uni-heidelberg.de (Dorn-Beineke, Alexandra)
Date: Thu, 20 Apr 2000 11:56:34 +0200
Subject: Cyclin D1
Message-ID: <4FDA723D969DD3118CBD009027B004290F03C9@Primnt.kli-ma>


Dear Collegues,
we are looking for a simple method to stain Cyclin-D1 for identification of
mantle cell lymphoma (translocation t11;14) by flow cytometry, ideally in
whole blood. Current protocols for cytoplasmic detection seem to fail and
also those published are difficult to reproduce and need significant amounts
of blood. As we get less and less volume of blood this becomes a problem.
Thanks for your support.

Alexandra Dorn-Beineke
Thomas Nebe

Dr. med. Alexandra Dorn-Beineke
F? f?r Laboratoriumsmedizin
Universit?tsklinikum Mannheim
Abt. H?matologie, Immunologie, Allergologie
Theodor-Kutzer-Ufer 1-3
D-68167 Mannheim
Tel: +49 621 383-3561 o. 3486
FAX: +49 621 383-3819
e-mail: alexandra.dorn-beineke at ikc.ma.uni.heidelberg.de
http://www.ma.uni-heidelberg.de/inst/ikc/





From djyoung at UCSD.Edu Wed Apr 19 16:04:02 2000
From: djyoung at UCSD.Edu (Dennis J. Young)
Date: Wed, 19 Apr 2000 14:04:02 -0700
Subject: Biotin-streptavidin staining
In-Reply-To: <38FCF61D.9C8BB9EF@garnet.tc.umn.edu>
Message-ID: <3.0.1.32.20000419140402.0083ce50@popmail.ucsd.edu>


At 06:56 PM 4/18/00 -0500, Kevin Waddick wrote:
>
> Is it really necessary to wash cells between incubation with a
>biotinylated antibody and adding streptavidin that is linked to a
>fluorochrome?
No. See:

Dale GL.
Rapid production of quasi-stable antibody-phycoerythrin conjugates for
use in flow cytometry.
Cytometry, 1998 Dec 1, 33(4):482-6.

> That is, doesn't it work as well if the two are added
>together, thereby saving a step? I suppose that I could test this
>myself, however I would consider limited attempts by me to be anecdotal.
>Others out there may have done actual studies of this and might even
>know of tricks to make it work -- assuming that it is not as simple as I
>described. That's what I'm hoping, anyway!
>
>Kevin G. Waddick, Ph.D.
>Parker Hughes Institute
>2657 Patton Road
>St. Paul, MN 55113
---
Dennis

Dennis J. Young
Mail:<<mailto:djyoung at ucsd.edu>>
WWW:<<http://cancer.ucsd.edu/SResources/flow.htm>>

Flow Cytometry Core Facility
University of California, San Diego
Internal Medicine Group, Bldg #4, Rm 126
9500 Gilman Drive La Jolla CA 92093-0671
Telephone:(858) 822-0407


From alberti at cmns.mnegri.it Wed Apr 19 15:54:53 2000
From: alberti at cmns.mnegri.it (Saverio Alberti)
Date: Wed, 19 Apr 2000 22:54:53 +0200 (MET DST)
Subject: mouse/human hepatocyte specific marker
In-Reply-To: <BCC4C6C4DF90D311B40A00A0C92C80270D0CFD@micro.microbio.uab.edu>
Message-ID: <Pine.OSF.3.96.1000419225216.587A-100000@alpha400.cmns.mnegri.it>


Trop-1 is expressed in fetal progenitor cells in human epidermis. This
may also be true for liver and othe epithelia.

Saverio Alberti
Head, Lab. of Experimental Oncology
Department of Cell Biology and Oncology
Consorzio Mario Negri Sud
66030 Santa Maria Imbaro (Chieti), Italy
Phone: (39-0872) 570.293
FAX: (39-0872) 570.412
E-mail: alberti at cmns.mnegri.it


On Wed, 19 Apr 2000, Dana Levasseur wrote:

>
> Hello All,
>
> Maybe this is a question the flow community can tackle? We are looking for
> an antibody specific for hepatocytes. Specifically, we are trying to
> differentiate between cells of erythroid versus other origins in fetal liver
> and were wondering if a "hepatocyte specific marker" existed. TER119 is the
> logical option for the erythroid marker. Also, I know the question was
> posed to the group a short time ago, but does there exist an erythroid
> marker that can differentiate more primitive precursors such as BFU-E,
> CFU-E, or normoblasts? Presumably, TER119 stains these cells, but it also
> stains retics. and mature erythrocytes.
>
> Many thanks in advance,
>
>
> Dana Levasseur
> University of Alabama at Birmingham
> Bevill Biomedical Research Building, Rm. 870
> 845 19th Street South
> Birmingham, AL 35205
>
> phone: (205)-934-1963
> e-mail: dnl at uab.edu
>



From thomasm at nshs.edu Wed Apr 19 12:19:06 2000
From: thomasm at nshs.edu (Tom Mc Closkey)
Date: Wed, 19 Apr 2000 13:19:06 -0400
Subject: A letter from AIDS Center
References: <38FB5331.8D35CB83@geo.net.ge>
Message-ID: <38FDEA8A.50499CF2@nshs.edu>




HIV/AIDS Patients Support Foundation - Georgia wrote:
>
> What is and how much is the prognostic importance of CD38 T cells
> subsets as activation markers in HIV infection.
>

CD8+CD38+ cells have been reported to add to the prognostic value of
CD4 cell counts in order to predict disease progression. This finding
is shown in:

Giorgi et al, Elevated levels of CD38+CD8+ T cell in HIV infection add
to the prognostic value of low CD4+ T cell levels: results of 6 years of
followup, J Acq Imm Def Syn, 6: 904-912, 1993.

Liu et al, Elevated relative fluorescence intesity of CD38 antigen
expression on CD8 T cells is a marker of poor prognosis in HIV
infection: results of 6 years of followup, Cytometry, 26: 1-7, 1996.

Tom

*****************************************************************************
Thomas W. Mc Closkey, Ph. D., Director of Flow Cytometry
North Shore University Hospital, New York University School of Medicine
Boas Marks Biomedical Research Center, 350 Community Drive
Manhasset, Long Island, New York 11030
ph: 516-562-4844 [office], 516-562-1135/4641 [lab] fax: 516-562-2866
*****************************************************************************


From thomasm at nshs.edu Wed Apr 19 12:04:12 2000
From: thomasm at nshs.edu (Tom Mc Closkey)
Date: Wed, 19 Apr 2000 13:04:12 -0400
Subject: EXPOv2 - EXPO32
References: <052EBD86E10ED41187870001FA7E1D0505B5DD@se-drc-mail2.draco.se.astra.com>
Message-ID: <38FDE70C.D645BC67@nshs.edu>




Marta.Borg at astrazeneca.com wrote:
>
> My question is, how can I know that EXPOv32 is better than EXPOv2 and why
> should I pay once more when I newer got what I paid for from the beginning,
> an adequate software? (And I made the mistake to by 12 copies!)
> Is there anyone out there who has the same problem?
>
> Please all EXPO users, speak up!
>

Last summer, wanting to upgrade our DOS based computer on the Elite, we
purchased a new EXPO workstation. We are also very disappointed with
this software [ver 2] as it is a step back in many regards from the DOS
based Elite software. When we communicated the deficiencies of this
software to Coulter we were told that a new version was being
developed. Still waiting. . .

Tom

*****************************************************************************
Thomas W. Mc Closkey, Ph. D., Director of Flow Cytometry
North Shore University Hospital, New York University School of Medicine
Boas Marks Biomedical Research Center, 350 Community Drive
Manhasset, Long Island, New York 11030
ph: 516-562-4844 [office], 516-562-1135/4641 [lab] fax: 516-562-2866
*****************************************************************************


From RFischer at therimmune.com Wed Apr 19 15:37:56 2000
From: RFischer at therimmune.com (Fischer, Randy)
Date: Wed, 19 Apr 2000 16:37:56 -0400
Subject: Biotin-streptavidin staining
Message-ID: <A01B29D8827CD311ADF10008C79F3DBF02B534@labsrv.therimmune.com>


Kevin,

In theory this does work, and it will work if you have carefully
titrated both the biotinylated antibody and streptavidin together. Many
commercial ELISA kits actually do this, and they work quite nicely and
reproducibly in my hands. As for doing the necessary work myself to get
the ratios correct, I think it would involve a lot of work and you would
need to repeat it every time you used a new batch of either the antibody
or the streptavidin. Howard probably knows someone who has done it
successfully and can tell you how much time it would take. Is it really
that much more time for one wash step?

Randy Fischer
TherImmune Research Corporation
9700 Great Seneca Hwy
Rockville, MD 20850
(240) 453-6256
RFischer at therimmune.com

> ----------
> From: Kevin Waddick
> Reply To: Kevin G Waddick
> Sent: Tuesday, April 18, 2000 4:56 PM
> To: Cytometry Mailing List
> Subject: Biotin-streptavidin staining
>
>
> Is it really necessary to wash cells between incubation with a
> biotinylated antibody and adding streptavidin that is linked to a
> fluorochrome? That is, doesn't it work as well if the two are added
> together, thereby saving a step? I suppose that I could test this
> myself, however I would consider limited attempts by me to be
> anecdotal.
> Others out there may have done actual studies of this and might even
> know of tricks to make it work -- assuming that it is not as simple as
> I
> described. That's what I'm hoping, anyway!
>
> Kevin G. Waddick, Ph.D.
> Parker Hughes Institute
> 2657 Patton Road
> St. Paul, MN 55113
>
>


From daviesd2 at icrf.icnet.uk Thu Apr 20 01:26:19 2000
From: daviesd2 at icrf.icnet.uk (Derek Davies)
Date: Thu, 20 Apr 2000 07:26:19 +0100 (BST)
Subject: Diagram to display fluorochrom emission overlap
In-Reply-To: <000701bfa9ba$e10e86a0$f767140a@ihi31382.imvs.sa.gov.au>
Message-ID: <Pine.SGI.3.96.1000420071055.710780A-100000@seagull.lif.icnet.uk>


On Wed, 19 Apr 2000, Joy Mundy wrote:
> Does anybody have a good diagram they can reference or send as an
> attachement that shows the spectral emission overlap for the following
> fluorchromes;
> FITC, PE, ECD and PCY5

Hi Joy,

Bio-Rad have, on their website, an excellent fluorochrome database that
allows you to plot emission and excitation spectra for a wide range of
fluorochromes:

http://fluorescence.bio-rad.com/

You will probably need to input ECD as Texas Red and PECy5 as Cy5 but it
should be able to give you an idea of the emissions and therfore the
overlap.

Other useful sites include:
Molecular Probes: http://www.probes.com/
Jerusalem College of
Technology: http://optics.jct.ac.il/~aryeh/Spectra/GIFs/

Derek

************************************************************************
Derek Davies Voice: (44) 0207 269 3394
FACS Laboratory, FAX: (44) 0207 269 3100
Imperial Cancer Research Fund, e_mail: derek.davies at icrf.icnet.uk
London, UK mobile: 07790 604112

Web Page: http://www.icnet.uk/axp/facs/davies/index.html

In tenebris lux
*************************************************************************





From miller at medicine.tamu.edu Thu Apr 20 08:14:40 2000
From: miller at medicine.tamu.edu (Jane S. Miller)
Date: Thu, 20 Apr 2000 08:14:40 -0500
Subject: macrophage apoptosis and flow
Message-ID: <s8febc8f.005@medicine.tamu.edu>


I'm wondering why you are using FL3 instead of FL2 for propidium iodine? Would that
effect the brightness?

>>> Maciej Simm <simmmmer at yahoo.com> 04/18/00 05:14PM >>>

Dear all,

One of the people I'm working with right now is studying apoptosis in
periteneal macrophages using a kit with annexin V monoclonal FITC ab
and propidium iodine.

The cells are autofluorescent. The "unstained" tube has signal in up
to 2nd log.

The stained tubes are VERY HIGHLY BRIGH on FL3 (PI) and I tried to
make things "fit" on the dotplot by lowering fl3 voltage by 200 or
so, but I'm still not getting everything (and also some of the dimmer
cells are squooshed on the axis). The FITC signal is of normal
brighness.

Needless to say we're not getting the same results as the kit
suggests we should given the incubation/conditions. He's gonna talk
to the company. I am posting this message with hopes that some of you
may have seen this method or one similar to it and are willing to
comment.

Any suggestions are welcome

thanks a bunch,

Maciej



=====
`---------------------------------------------`
| Maciej S. Simm | 525 E 68th Street |
| Research Technician | Room N-805 |
| Cornell Medical Center | Tel. 212.746.3428 |
`---------------------------------------------`
| www.cd4cd8.com |
`---------------------------------------------`

__________________________________________________
Do You Yahoo!?
Send online invitations with Yahoo! Invites.
http://invites.yahoo.com



From bourin at attglobal.net Wed Apr 19 11:42:27 2000
From: bourin at attglobal.net (bourin)
Date: Wed, 19 Apr 2000 18:42:27 +0200
Subject: Cord Blood Cell Fixation
References: <1a.18491c6.2611048d@aol.com>
Message-ID: <38FDE1F2.7E1AAE7C@attglobal.net>

You could not appreciate cell viability after fixation. It is unclear if you lyse
red blodd cells or not. If not, you must do it. Try formol (1% in PBS), it works
well for a 3 to 4 days delay. For more, try Stabilcyte from BioE.

MSchwa3722 at AOL.COM wrote :

> Does anyone have a simple protocol for staining and fixing whole cord blood
> for later acquisition. The samples I have run have shown significantly
> decreased viability after fixation and delayed acquisition. We determine the
> percentage of CD3 and CD34 positive cells based on the entire WBC population.
> I suspect the cord blood granulocytes in the sample are contributing to the
> decreased viability but due to the purpose of fixation for later acquisition
> I would prefer not to have to ficoll the samples prior to staining. Perhaps
> this is my only alternative.
>
> If anyone has any suggestions that may help I'd appreciate hearing from you.
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From vannacci at pharm.unifi.it Thu Apr 20 02:45:14 2000
From: vannacci at pharm.unifi.it (Alfredo Vannacci)
Date: Thu, 20 Apr 2000 09:45:14 +0200
Subject: Thanks for basophils!
Message-ID: <000201bfaa9c$5db61f60$9745d996@pharm.unifi.it>

Dear FLOWers,
I wish to thank everybody who answered my question about gating basophils.
You've been precious.

Dr Alfredo Vannacci, MD

Universit? degli Studi di Firenze
Dipartimento di Farmacologia Preclinica e Clinica "Mario Aiazzi Mancini"
Unit? Operativa di Tossicologia Medica e Medicina delle
Farmacotossicodipendenze
Viale Pieraccini 6, 50139, Firenze, Italia

e-mail vannacci at pharm.unifi.it

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From joseph.beckham at usa.net Wed Apr 19 12:33:07 2000
From: joseph.beckham at usa.net (Joseph Beckham)
Date: Wed, 19 Apr 2000 12:33:07 -0500
Subject: Employment Position Available
Message-ID: <a04310100b5239dafee21@[129.112.21.8]>

Staff Scientist - Flow Cytometry
Intramural Research Program
National Institute of Arthritis and Musculoskeletal
and Skin Diseases
National Institutes of Health

The National Institute of Arthritis and Musculoskeletal and Skin
Diseases (NIAMS) of the National Institutes of Health (NIH) is
seeking exceptional candidates to fill the position of Staff
Scientist - Flow Cytometry in the Autoimmunity Branch of the
Intramural Research Program (IRP) of NIAMS, NIH. The successful
candidate will manage a newly established flow cytometry laboratory
that has a 3 laser FACSVantage with turbo sort for sorting and a
FACcaliber for analysis and all supporting equipment, computers and
software. Plans to add a Cytomation MoFlo high-speed sorter are
being developed. The individual will be responsible for managing and
organizing the laboratory and the design of specific projects.
Candidates must have either an M.D. or Ph.D. degree and professional
knowledge and experience in contemporary flow cytometry sufficient to
independently organize and conduct clinical and basic immunologic
research using this technology. Applicants should submit their
curriculum vitae and bibliography to:

Dr. Peter Lipsky
c/o Scott Sigley, NIAMS, HRMB
Building 31, Room 4C13
31 Center Drive, MSC 2350
Bethesda, MD 20892-2350
or e-mail ss403p at nih.gov

Applications must be postmarked by 5/15/00.

NIH is an Equal Opportunity Employer
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From enockson at musc.edu Wed Apr 19 10:02:51 2000
From: enockson at musc.edu (Candace Enockson)
Date: Wed, 19 Apr 2000 11:02:51 -0400
Subject: unexpected T and B phenotypes after culture
In-Reply-To: <B5213E3A.7A9%kbahjat@ufl.edu>
Message-ID: <274543.3165130971@[128.23.89.125]>


Keith, are you not able to gate these cells out based on their scatter -
lower FSC and higher SSC - or are they contaminating your population of
interest?
Candace Enockson

--On Mon, Apr 17, 2000 10:18 PM -0400 Keith Bahjat <kbahjat at ufl.edu> wrote:

>
> I can say that in our culture systems following activation, we often find
> cells of questionable viability which will bind any antibody you throw in
> the tube. You need to make sure you have some marker that these cells are
> negative for, like CD13, or F4/80. Something you would never expect to
> find on a T cell. When you're in the habit of staining with 4 antibodies,
> and looking for cells positive for all 4 antibodies, you're just asking
> for trouble.
> kb
>
> Keith Bahjat
> kbahjat at ufl.edu
>
>
>
> on 4/14/00 10:03 PM, Qi, Hai at haqi at utmb.edu wrote:
>
>>
>> It is not surprising. I believe you will see double positive cells, and
>> they are primarily ACTIVATED T cells (this is potentially why you find
>> they are bigger, assuming not too many doublets are there). Further, you
>> may see T cells expressing different levels of B220. In my experience,
>> this phenomenon is also not specific to cells in culture. Overall, I
>> don't think B220 is really an absolute marker for B cells. Hopefully
>> experts out there will comment on this.
>>
>> To gate out cell aggregates, I think you can plot pulse height vs width
>> of your CD4/CD8 channel (several washes with ice-cold staining buffer
>> should also help).
>>
>> Hai Qi
>> Pathology, UTMB
>>
>> -----Original Message-----
>> From: Olindo Assis [mailto:oamfilho at cpqrr.fiocruz.br]
>> Sent: Wednesday, April 12, 2000 1:01 PM
>> To: Cytometry Mailing List
>> Subject: unexpected T and B phenotypes after culture
>>
>>
>> We have been phenotyping splenocytes from different strains
>> of mouse before
>> and after stimulation with bacteria (H. pylori) antigens. We
>> are basically
>> labeling the cells using anti-CD4, anti-CD8 and
>> anti-CD45(B220) PE from
>> SIGMA. Labeling procedures were done in separated tubes.
>> Frequently we have noticed that the sum of CD4 + CD45 + CD8
>> cells from the
>> unstimulated cultures give us a number over 100%.
>> It is interesting to observe that this phenomena is observed
>> only in
>> unstumulated cells. Cultures in the presence of H. pylori
>> antigens does not
>> show this problem. Moreover it was observed for all mouse
>> strains evaluated
>> independent of the age. We have used Balb/C, C3H/HeN and
>> C57/BL6.
>> It is interesting to observe that these overestimation is
>> observed only in
>> the region corresponding to larger cells. We first suggested
>> that this could
>> be due to the presence of doublets of CD4 and CD45 cells.
>> However it could
>> be also due to the presence of cells co-expressing both
>> phenotypes.
>> In order to solve this we are currently evaluating the
>> presence of double
>> labeled cells using anti-CD4 FITC and anti-CD45 PE in the
>> same tube.
>> However, if we find double labeled cells we still have the
>> question whether
>> they are representing doublets or bi-phenotypic cells.
>> Does any one have experience of phenotypic analysis of T and
>> B splenocytes
>> after control cultures? Any suggestions?
>> Is that possible that CD45 (B220) could be a marker for
>> cultured CD4 cells?
>>
>>
>> Any help is appreciated,
>>
>> Olindo
>>
>






From lwarma at med.unc.edu Wed Apr 19 17:57:32 2000
From: lwarma at med.unc.edu (Larry Arnold)
Date: Wed, 19 Apr 2000 17:57:32 -0500
Subject: DAPI and APC on 2nd laser lines
In-Reply-To: <17774C9FD541D211A95800805FE6AE9B01F0749E@US4N65>
Message-ID: <200004192155.RAA29988@redtail.med.unc.edu>


Sam

As a general rule I always try to reflect the dimmest signal and pass the
brighter signal. The loss is most significant when passing thru a filter.

Larry

At 07:50 PM 4/18/00 -0400, you wrote:
>
>Hi Group
> I'm trying to collect DAPI and APC signals on the second (and third)
>laser lines of a FACStar Plus in a manner very similar to that described by
>Moore et al in:
> Cytometry (32) 1: 57-65 , May 1998. The method describes the
>collection of DAPI and APC fluorescence from cells excited by collinear
>beams of a mixed gas laser for UV (350-356 nm) and a HeNe laser (633 nm).
>
>In this technical note, the filters adjacent to the relevant PMT's are
>described (660DF20 for APC and 450DF20 for DAPI), but the splitter between
>the two PMT's is not mentioned.
> I have looked around and cannot find a reference as to the "proper
>thing to do" in this case.
>
> The DAPI signal is very bright... should I reflect it and filter the
>APC fluorescence or should I filter the DAPI and reflect the APC?
>
>Is there a general rule that I could apply based on wavelength regardless of
>"brightness"?
>
> Thanks in Advance,
>
> I look forward to your input.
>
> Cheers,
> Sam
>Sam Witherspoon
>sw11527 at glaxowellcome.com
>
>Dept. of Receptor Biochemistry Tel. 919-483-3078
>Glaxo Wellcome R&D Page 919-857-7768
>5 Moore Dr. Fax 919-483-0585
>RTP, NC 27709
>
Larry W. Arnold, Ph.D.
Res. Assoc. Prof.
Director, Flow Cytometry Facility
Department of Microbiology and Immunology
CB# 7290
University of North Carolina
Chapel Hill, NC 27599
Phone: 919-966-1530
FAX: 919-962-8103


From martha.siegel at quintiles.com Thu Apr 20 09:57:08 2000
From: martha.siegel at quintiles.com (martha.siegel@quintiles.com)
Date: Thu, 20 Apr 2000 10:57:08 -0400
Subject: Flow meeting
Message-ID: <852568C7.005234C7.00@qrtplc01.qrtp.quintiles.com>

(See attached file: Flow Meeting - April 24th.doc)
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From Howard.Robinson at joslin.harvard.edu Fri Apr 21 09:56:18 2000
From: Howard.Robinson at joslin.harvard.edu (Robinson, Howard)
Date: Fri, 21 Apr 2000 10:56:18 -0400
Subject: Seeking Shareware/Freeware for Cell Cycle Analysis
Message-ID: <047232CBD5F8D21194240008C7F3625804D681DC@josmail.harvard.edu>


Hi,

I'm having trouble locating any Shareware/Freeware for Cell Cycle Analysis.
I was wondering if anyone is using any that they could tell me about, or if
anyone has even heard about any PC based Shareware/Freeware.

Thanks very much,

Howard Robinson


From butterfield.15 at osu.edu Thu Apr 20 14:41:10 2000
From: butterfield.15 at osu.edu (Amy Butterfield)
Date: Thu, 20 Apr 2000 15:41:10 -0400
Subject: mitochondrial probes
Message-ID: <v03130300b5250ca35aa1@[164.107.3.187]>


If anyone has used the mitochondrial membrane probes such as JC-1 or
MitoTracker can you tell me what works as a positive control?

Thanks.




From simmmmer at yahoo.com Thu Apr 20 16:08:08 2000
From: simmmmer at yahoo.com (Maciej Simm)
Date: Thu, 20 Apr 2000 14:08:08 -0700 (PDT)
Subject: putting surface markers in fixed cells
Message-ID: <20000420210808.13837.qmail@web2003.mail.yahoo.com>


antigen retrieval might also work on these cells. I lost the original
email address of the person who posted it, but here are references
for AR:


John: Here are a few pertinent references:

1. Shi,S. et al. Antigen retrieval in formalin-fixed
parrafin-embedded tissues:....... J.Histchem.Cytochem.39:741-748
(1991).

2. Taylor,CR et al. Strategies for improving the immunohistochemical
staining of... Hum.Pathol.25:263-270 (1990).

3. Momose H. et al. Antigen retrieval by microwave irradiation in
lead thiocyanate.... Appl.Immunohistochem.1:77-82 (1993).

4.Leong,A.S-Y. & J. Milios An assessment of the efficacy of the
microwave antigen-retrieval... Appl.Immunohistochem.1:267-274 (1993).

5. Cuevas,E.C. et al. Microwave antigen retrieval in
immunocytochemistry: A study of 80 Abs. J.Clin.Pathol.47:448-452
(1994).

6. Gown,A.M. et al. Microwave-based antigenic unmasking......
Appl.Immunohistchem.1:256-266 (1993).



as far as flozen tissue slides I've worked with it involves simmering
the specimen in citrate buffer using microwave oven. Not friendly to
morphology.

=====
`---------------------------------------------`
| Maciej S. Simm | 525 E 68th Street |
| Research Technician | Room N-805 |
| Cornell Medical Center | Tel. 212.746.3428 |
`---------------------------------------------`
| www.cd4cd8.com |
`---------------------------------------------`

__________________________________________________
Do You Yahoo!?
Send online invitations with Yahoo! Invites.
http://invites.yahoo.com


From thomasm at nshs.edu Thu Apr 20 15:46:50 2000
From: thomasm at nshs.edu (Tom Mc Closkey)
Date: Thu, 20 Apr 2000 16:46:50 -0400
Subject: DAPI and APC on 2nd laser lines
References: <17774C9FD541D211A95800805FE6AE9B01F0749E@US4N65>
Message-ID: <38FF6CBA.EA56B529@nshs.edu>


"Witherspoon, Sam" wrote:
>
> I'm trying to collect DAPI and APC signals on the second (and third)
> laser lines of a FACStar Plus
> The DAPI signal is very bright... should I reflect it and filter the
> APC fluorescence or should I filter the DAPI and reflect the APC?

Hi Sam,

I recently tried to work this combo out on our Elite. Setup was 488
beam displaced from the other two beams [uv and 633] which were set
colinear. An unexpected result was that DAPI fluorescence showed up in
the APC signal. I tried different bandpass and longpass filters for the
APC and ran samples with variuos lasers blocked to verify the souurce of
the signal. My conclusion was that DA{PI simply fluoresces way out into
the red, at least under the experiemental conditions I used.

Regards,
Tom



*****************************************************************************
Thomas W. Mc Closkey, Ph. D., Director of Flow Cytometry
North Shore University Hospital, New York University School of Medicine
Boas Marks Biomedical Research Center, 350 Community Drive
Manhasset, Long Island, New York 11030
ph: 516-562-4844 [office], 516-562-1135/4641 [lab] fax: 516-562-2866
*****************************************************************************


From tylee at itis.com Thu Apr 20 19:33:45 2000
From: tylee at itis.com (tylee)
Date: Thu, 20 Apr 2000 19:33:45 -0500
Subject: Fluorochrom Spectra Software Inquiry
Message-ID: <01bfab29$40d87680$7bbda5d8@triss2>


Dear "Fluoresecent" Chemists,

I have a question related to the inquiry posted earlier by Joy...

Does anyone know of a program (software, etc) that will plot emission data
so it shows up as the appropriate color corresponding to the wavelength on
the X-axis? Can a macro be incorporated into Excell to do that???
If I remember correctly, I think BioRads site shows the same color for the
line all the way through the excitation or emission spectrum. I would like
to be able to show the color change depending on the wavelength.

Ty Lee



-----Original Message-----
From: Joy Mundy <joy.mundy at imvs.sa.gov.au>
To: cyto-inbox
Date: Wednesday, April 19, 2000 4:11 PM
Subject: Diagram to display fluorochrom emission overlap


>
>HI,
>
>Does anybody have a good diagram they can reference or send as an
>attachement that shows the spectral emission overlap for the following
>fluorchromes;
>
>FITC, PE, ECD and PCY5
>
>
>Thanks
>
>Joy Mundy
>Division of Human Immunology
>I.M.V.S.
>PO Box 14
>Rundle Mall PO
>Adelaide 5000
>Australia
>
>joy.mundy at imvs.sa.gov.au
>
>Phone (08) 8222-3476
>Fax (08) 8232-4092
>
>



From adwells at mail.MED.UPENN.EDU Thu Apr 20 13:52:43 2000
From: adwells at mail.MED.UPENN.EDU (Andrew D. Wells)
Date: Thu, 20 Apr 2000 14:52:43 -0400
Subject: internal staining for p27kip
Message-ID: <B524CA3B.CA8%adwells@mail.med.upenn.edu>


speaking of p21 staining, has anyone had success achieving good internal
staining for p27kip? My search of the literature has not been successful.
Any clones, protocols or ref's would be appreciated


Andrew D. Wells, Ph.D.
University of Pennsylvania
Department of Medicine
728 Clinical Research Building
415 Curie Boulevard
Philadelphia, PA 19104

(215) 573-1840 [office]
(215) 898-1951 [laboratory]
(215) 573-2880 [FAX]

adwells at mail.med.upenn.edu



From thomasm at nshs.edu Thu Apr 20 15:32:51 2000
From: thomasm at nshs.edu (Tom Mc Closkey)
Date: Thu, 20 Apr 2000 16:32:51 -0400
Subject: macrophage apoptosis and flow
References: <20000418221426.1357.qmail@web2004.mail.yahoo.com>
Message-ID: <38FF6973.8AA83326@nshs.edu>


Maciej Simm wrote:
>
> One of the people I'm working with right now is studying apoptosis in
> periteneal macrophages using a kit with annexin V monoclonal FITC ab
> and propidium iodine.
>
> The cells are autofluorescent. The "unstained" tube has signal in up
> to 2nd log.
>

If you're having trouble with high autofluroescence, you';ll do well to
move away from the green and into the red part of the spectrum. Annexin
APC would be a better choice for example. Or an alternative assay may
be more appropriate: TUNEL biotin with a red avidin.

As an aside, we recently observed very high levels of constitutive
annexin binding to freshly isolated monocytes. We didn't follow up on
this as we were interested in lymphocyte death and are not sure of its
significanece.

I strongly recommend using DNA staining and fluorescence microscopy to
verify you r flow assay for apoptosis.

Good luck,
Tom

*****************************************************************************
Thomas W. Mc Closkey, Ph. D., Director of Flow Cytometry
North Shore University Hospital, New York University School of Medicine
Boas Marks Biomedical Research Center, 350 Community Drive
Manhasset, Long Island, New York 11030
ph: 516-562-4844 [office], 516-562-1135/4641 [lab] fax: 516-562-2866
*****************************************************************************


From bunny at cotleur.com Thu Apr 20 21:30:37 2000
From: bunny at cotleur.com (bunny)
Date: Thu, 20 Apr 2000 22:30:37 -0400
Subject: mea culpa
Message-ID: <38FFBD47.417C12DA@cotleur.com>


Well, there ain't nothing like embarassing yourself in front of the masses!
Re: the weird "quenching" I inquired on, I have been reminded that PERCP
canNOT bleed into Fl2; therefore not only is compensation unnecessary,
but the effect we observed was really in the SINGLE stain tube (and not
a result of the additional Ab). When the second, increasingly diluted
Ab was added- the odd +/+ population shifted back to the normal +/-
[quadrant 1] position.
My colleagues are retesting several donors to determine if this was a
one-time, bad prep or if the particular donor has some sort of
interfering factor that causes CD4-Percp stained cells to fluoresce
bright Fl3/2.

Embarassment aside- a fresh view is ALWAYS welcome and I cannot rave
enough about the help I have received from this group!
Happy Easter-from the real thing!
--



Bunny

PS- to those that "just had to ask"; I was named this at birth- by
parents who CLEARLY did not give enough thought to a future with this
moniker! :-)
<<ok, maybe it was after one to many beers..>>








*******************************************************
Bunny Cotleur +*+ Bunny Cotleur
Cleveland Clinic Foundation *+* 2001 Lester RD
Neurosciences NC30 +*+ Valley City, OH 44280
9500 Euclid Avenue *+* 330-483-4800
Cleveland, OH 44195 +*+ bunny at cotleur.com
216-444-1164 *+*
cotleua at ccf.org +*+

*******************************************************
When you do something, you should burn yourself completely, like a good
bonfire, leaving no trace of yourself.
(Shunryu Suzuki)


From DARZYNK at nymc.edu Thu Apr 20 16:52:08 2000
From: DARZYNK at nymc.edu (DARZYNKIEWICZ ZBIGNIEW)
Date: Thu, 20 Apr 2000 17:52:08 -0400
Subject: G0 cells,
Message-ID: <8C3E01568EC0D311AF0600508B0CC32B020F19E5@MAIL>


The term G0 was introduced over 35 years ago in operational sense, to define
cells that do not enter the cycle (incorporate thymidine) for a time period
equivalent of two generation times.This term was then adapted by many
researchers to define noncycling cells in general or in their particular
cell systems, often without any specification. There is still no general
consensus as to which attribute of the cell should be considered the marker
of Go. Years ago we have noticed that noncycling cells have approximaletely
10 times less RNA than their cycling counterparts (PNAS, 73:2881-2884, 1976)
. For many years the low RNA content was considered to be the gold standard
of Go state. Cell staining with the metachromatic dye acridine orange or
Hoechst/pyronine Y allows one to obtain differential staining of DNA and RNA
and easily identify Go cells (e.g see Current Protocols in Cytometry). Low
mitochondrial mass and overall mitochondrial transmembrane potential (e.g.
measured by rhodamine 123 uptake) was later found to be another marker of Go
cells (PNAS, 78: 2383-2387, 1981).
With molecular markers of the cell cycle now available the Go state can be
defined more specifically than it was possible before. I would consider the
cells to be in Go if they still did not yet phosphorylate pRB (prior to the
"restriction point"). The antibodies that sense pRB phosphorylation are
available and can be used to identify such cells (e.g. Juan et al., Exp.
Cell Res, 239: 104-110, 1998). One can also define Go cells as the
postmitotic cells that did not yet express D type cyclins (Cytometry, 25:
1-13, 1996). It should be noted, however, that the cell ability to enter
genuine Go characterizes normal cells. Most tumor type cells have either
defective pRB pathway or constant mitogenic signaling upstream of pRB and
therefore by-pass Go.
Zbigniew Darzynkiewicz


From stetler at box-s.nih.gov Fri Apr 21 09:45:57 2000
From: stetler at box-s.nih.gov (Maryalice Stetler-Stevenson)
Date: Fri, 21 Apr 2000 10:45:57 -0400
Subject: Fwd: Cyclin D1
Message-ID: <v04220800b526189b6979@[156.40.224.68]>


We worked on this and found the detection of cyclin D1 to be highly
technique dependent. You can not viably freeze the cells, exact
procedures must be followed and someone having a bad day- a tiny bit
off in timing, etc- can't reproduce this test. Matter of fact, only
one person in the lab could reproduce this test. Surprisingly, we
found that DNA content analysis was as good at detecting mantle cell
lymphoma and much more dependable a method. Since the primary
differential diagnosis is between CLL and mantle cell and since CLL
doesn't cycle and mantle cell does, you can make a diagnosis. I have
talked to many who have tried and come to the same conclusion- don't
try to use cyclin D1 in the clinical lab. It is a waste of your time.


Maryalice


>From: "Dorn-Beineke, Alexandra"
><alexandra.dorn-beineke at ikc.ma.uni-heidelberg.de>
>To: Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu>
>Subject: Cyclin D1
>Date: Thu, 20 Apr 2000 11:56:34 +0200
>X-PMFLAGS: 34078848 0 1 9869.CNM
>
>
>Dear Collegues,
>we are looking for a simple method to stain Cyclin-D1 for identification of
>mantle cell lymphoma (translocation t11;14) by flow cytometry, ideally in
>whole blood. Current protocols for cytoplasmic detection seem to fail and
>also those published are difficult to reproduce and need significant amounts
>of blood. As we get less and less volume of blood this becomes a problem.
>Thanks for your support.
>
>Alexandra Dorn-Beineke
>Thomas Nebe
>
>Dr. med. Alexandra Dorn-Beineke
>F? f?r Laboratoriumsmedizin
>Universit?tsklinikum Mannheim
>Abt. H?matologie, Immunologie, Allergologie
>Theodor-Kutzer-Ufer 1-3
>D-68167 Mannheim
>Tel: +49 621 383-3561 o. 3486
>FAX: +49 621 383-3819
>e-mail: alexandra.dorn-beineke at ikc.ma.uni.heidelberg.de
>http://www.ma.uni-heidelberg.de/inst/ikc/

Maryalice Stetler-Stevenson
Director Flow Cytometry Unit
Laboratory of Pathology, NCI, NIH


From thomas at tritechinc.com Fri Apr 21 09:14:51 2000
From: thomas at tritechinc.com (Joanne Thomas)
Date: Fri, 21 Apr 2000 10:14:51 -0400
Subject: CD11b expression on Lymphocytes
Message-ID: <008b01bfab9b$f5f025a0$1801a8c0@jill.tritechinc.com>


Maciej:
CD11a is expressed on all leukocytes, CD11b &c are expressed on monocytes,
granulocytes and NK cells. The CD11's are the alpha chains of beta2 integrin
family while CD18 represents the beta chain. CD11b is used most often
because it is the most densely expressed on the cell surface (i.e.
brightest) and has the largest increase upon stimulation. You don't mention
any stimulation of your cells in your procedure, we used 60ng/mL (final
concentration) in a whole blood assay. (See O'Gorman et al; Annls NY Acad
Sci, 1993; 667:427-430). We once had a patient that had decreased CD11b on
resting but normal levels upon stimulation. This child had mild disease.
Also, are you gating on lymphocytes as your title suggests? You should
really be gating on your granulocytes for CD11b. Since CD11b and CD18
represent different chains of the beta2 integrin, it is feasible to have a
defect in one and not the other. I don't know what the clinical significance
would be. Any comments Dr. O'Gorman??

Joanne Thomas, M.S.
Director of Operations
TRITECH Field Engineering
2014 Renard Court, Suite I
Annapolis, MD 21401
1-800-886-7004 (USA)
1-410-266-1522
410-266-0935 (FAX)
thomas at tritechinc.com
-----Original Message-----
From: Maciej Simm <simmmmer at yahoo.com>
To: cyto-inbox
Date: Thursday, April 20, 2000 4:55 PM
Subject: CD11b expression on Lymphocytes


>
>Greetings once again,
>
>We have recently received a blood specimen and were asked to stain
>for CD18/11b ( I love it how doctors don't explain the condition of
>the patient ahead of time, so we know what to look for )
>
>We found, that when compared to our NC's (N=300+ for this "tube" and
>growing) this patient had less +/+ lymphoctes (~30% vs. 12%). The
>expression of CD18 was not reduced - all of his leukocytes had >97%.
>
>A question came up: what is the significance of CD18+/CD11b-
>lymphocytes? what is the significance of a decrease in the +/+
>population?
>
>We do not routinely do CD11a or CD11c - are these mutually exclusive?
>I.e. it's either a, b or c but never any of them together?
>
>And finally, is the ligand for CD11b soluble and may have interfered
>with our staning?
>
>This staining was done in whole blood (which I just found is a
>no-no).
>
>I would appreciate if anyone could share any thoughts on this topic.
>
>Maciej
>
>__________________________________________________
>Do You Yahoo!?
>Send online invitations with Yahoo! Invites.
>http://invites.yahoo.com
>



From kbahjat at ufl.edu Thu Apr 20 16:19:12 2000
From: kbahjat at ufl.edu (Keith Bahjat)
Date: Thu, 20 Apr 2000 17:19:12 -0400
Subject: CD11b expression on Lymphocytes
In-Reply-To: <20000419213735.22592.qmail@web2003.mail.yahoo.com>
Message-ID: <B524EC8F.BF5%kbahjat@ufl.edu>


Do a medline search for Leukocyte Adhesion Deficiency-1, or LAD-1. A
mutation in the CD18 locus on chromosome 21 causes LAD-1. CD18 is the shared
beta subunit of three different adhesion heterodimers, LFA-1 on B,T, and NK
cells; CR3 (or CD11b) on PMNs, momos, macs, eos, and NK cells; and p150,95,
another complement receptor. Delayed separation of the umbilical cord is
usually the first sign. patients have significantly elevated PMN counts
without infection.

You should have been looking for CD11b expression within the granulocytic
populations, not the lymphs. The CD11b positive lymphs were NK cells. the
difference in % may be due to normal variation in NK cell numbers amongst
the population. The CD18 expression on the lymphs is enough to rule out
LAD-1, I believe.

I love what physicians do with our health care dollars sometimes......

kb

--
Keith Bahjat
Graduate Assistant
University of Florida
College of Medicine
Gainesville, Florida
Voice: (352) 392-4887
Fax: (352) 392-5393
e-mail: kbahjat at ufl.edu


> From: Maciej Simm <simmmmer at yahoo.com>
> Date: Wed, 19 Apr 2000 14:37:35 -0700 (PDT)
> To: Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu>
> Subject: CD11b expression on Lymphocytes
>
>
> Greetings once again,
>
> We have recently received a blood specimen and were asked to stain
> for CD18/11b ( I love it how doctors don't explain the condition of
> the patient ahead of time, so we know what to look for )
>
> We found, that when compared to our NC's (N=300+ for this "tube" and
> growing) this patient had less +/+ lymphoctes (~30% vs. 12%). The
> expression of CD18 was not reduced - all of his leukocytes had >97%.
>
> A question came up: what is the significance of CD18+/CD11b-
> lymphocytes? what is the significance of a decrease in the +/+
> population?
>
> We do not routinely do CD11a or CD11c - are these mutually exclusive?
> I.e. it's either a, b or c but never any of them together?
>
> And finally, is the ligand for CD11b soluble and may have interfered
> with our staning?
>
> This staining was done in whole blood (which I just found is a
> no-no).
>
> I would appreciate if anyone could share any thoughts on this topic.
>
> Maciej
>
> __________________________________________________
> Do You Yahoo!?
> Send online invitations with Yahoo! Invites.
> http://invites.yahoo.com



From xyinc3 at frii.com Fri Apr 21 20:20:11 2000
From: xyinc3 at frii.com (Mike Evans)
Date: Fri, 21 Apr 2000 19:20:11 -0600
Subject: FW: macrophage apoptosis and flow
Message-ID: <LNBBJBKBINLMKMFCGIJMCECACDAA.xyinc3@frii.com>




-----Original Message-----
From: Mike Evans [mailto:xyinc3 at frii.com]
Sent: Friday, April 21, 2000 7:20 PM
To: cyto-inbox
Subject: RE: macrophage apoptosis and flow


Normally Pmt's are not very responsive at the voltages you spoke of and will
not be linear as well. What is the filter you are using for the PI. Is it
a long pass. If it is you are going to get loads of light. Maybe you can
put in a Band Pass filter with a fairly tight bandwidth. I would recommend a
620/20 filter for the PI. This should drop the overall intensity enough
that it should allow you to run under linear conditions as well as get
everything that should be on scale on scale.

Mike Evans

-----Original Message-----
From: Jane S. Miller [mailto:miller at medicine.tamu.edu]
Sent: Thursday, April 20, 2000 7:15 AM
To: cyto-inbox
Subject: Re: macrophage apoptosis and flow



I'm wondering why you are using FL3 instead of FL2 for propidium iodine?
Would that
effect the brightness?

>>> Maciej Simm <simmmmer at yahoo.com> 04/18/00 05:14PM >>>

Dear all,

One of the people I'm working with right now is studying apoptosis in
periteneal macrophages using a kit with annexin V monoclonal FITC ab
and propidium iodine.

The cells are autofluorescent. The "unstained" tube has signal in up
to 2nd log.

The stained tubes are VERY HIGHLY BRIGH on FL3 (PI) and I tried to
make things "fit" on the dotplot by lowering fl3 voltage by 200 or
so, but I'm still not getting everything (and also some of the dimmer
cells are squooshed on the axis). The FITC signal is of normal
brighness.

Needless to say we're not getting the same results as the kit
suggests we should given the incubation/conditions. He's gonna talk
to the company. I am posting this message with hopes that some of you
may have seen this method or one similar to it and are willing to
comment.

Any suggestions are welcome

thanks a bunch,

Maciej



=====
`---------------------------------------------`
| Maciej S. Simm | 525 E 68th Street |
| Research Technician | Room N-805 |
| Cornell Medical Center | Tel. 212.746.3428 |
`---------------------------------------------`
| www.cd4cd8.com |
`---------------------------------------------`

__________________________________________________
Do You Yahoo!?
Send online invitations with Yahoo! Invites.
http://invites.yahoo.com



From mogorman at nwu.edu Fri Apr 21 09:09:23 2000
From: mogorman at nwu.edu (Maurice R.G. O'Gorman)
Date: Fri, 21 Apr 2000 08:09:23 -0600
Subject: CD11b expression on Lymphocytes
Message-ID: <v02130500b5260ed1f7fe@[10.1.200.158]>


Hello Maciej

it is most likely that your physician was querying a diagnosis of
"Leukocyte Adhesion Deficiency Type-1". This is a disorder caused in every
case identified to date by mutations in the Beta chain (CD18). Mutations
in CD18 lead to the abnormal expression of all of the beta 2 leukocyte
integrins (CD11a, CD11b and CD11c), since the proper expression of CD18 is
required for their normal expression. Clinically patients suffer from
severe difficult to treat non-purulent skin lesions, gingivitis and
periondontitis. The common clinical condition cited is delayed umbilical
cord separation. The most facile method for detecting LAD-1 is to gate on
neutrophils and look for the expression of CD11b on both resting and in
vitro activated whole blood. Yes this can be done in whole blood. We have
developed a diagnostic test which is extrely easy and can be performed in
only a few hours. Please refer to "O'Gorman, McNally, Andeson and Myones.
A rapid whole blood lysis technique for the diagnosis of moderate or severe
Leukocyte Adhesion Deficiency Type-1. Annal. NY Acad. Sci.. 677:427-430,
1993. We offer this test on a routine basis in our laboratory and would be
happy to provide you with a NCCLS formatted laboratory procedure.

Regards

Mo.

>Greetings once again,
>
>We have recently received a blood specimen and were asked to stain
>for CD18/11b ( I love it how doctors don't explain the condition of
>the patient ahead of time, so we know what to look for )
>
>We found, that when compared to our NC's (N=300+ for this "tube" and
>growing) this patient had less +/+ lymphoctes (~30% vs. 12%). The
>expression of CD18 was not reduced - all of his leukocytes had >97%.
>
>A question came up: what is the significance of CD18+/CD11b-
>lymphocytes? what is the significance of a decrease in the +/+
>population?
>
>We do not routinely do CD11a or CD11c - are these mutually exclusive?
>I.e. it's either a, b or c but never any of them together?
>
>And finally, is the ligand for CD11b soluble and may have interfered
>with our staning?
>
>This staining was done in whole blood (which I just found is a
>no-no).
>
>I would appreciate if anyone could share any thoughts on this topic.
>
>Maciej
>
>__________________________________________________
>Do You Yahoo!?
>Send online invitations with Yahoo! Invites.
>http://invites.yahoo.com

********************************************
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
********************************************
Dr. Maurice (Mo) R.G. O'Gorman
Associate Professor Pediatrics
Northwestern Univ. Med. School
Director Diagnostic Immunology and Flow Cytometry Laboratories
The Children's Memorial Hospital
2300 Children's Plaza
Chicago, IL 60614
Ph. 773 880 3070 office
773 880 4361 laboratory
Fax 773 880 3739




From qnguyen at mail.ahc.umn.edu Sat Apr 22 19:24:53 2000
From: qnguyen at mail.ahc.umn.edu (Quang Nguyen)
Date: Sat, 22 Apr 2000 19:24:53 -0500
Subject: Avian Rbc cell lysis
Message-ID: <390242D5.A4665915@lenti.med.umn.edu>


I was wondering if the avian RBC lysis was critical whie looking for
the lymphocyte population



From martha.siegel at quintiles.com Fri Apr 21 11:12:34 2000
From: martha.siegel at quintiles.com (martha.siegel@quintiles.com)
Date: Fri, 21 Apr 2000 12:12:34 -0400
Subject: Flow Meeting
Message-ID: <852568C8.00590794.00@qrtplc01.qrtp.quintiles.com>

Sorry about attachment. Here it is again.



Announcing
-------------- next part --------------

?.. the 2nd meeting of the
Greater Atlanta Flow Cytometry Group

Sponsor: CompuCyte Corporation, Don Cornelius

Steering Committee: Martha Siegel, Diane Hanf, Astrid Ortiz
Quintiles Laboratories, Ltd., Smyrna, Georgia

When: Monday, April 24th 1:00 p.m. - 4:30p.m.

Where: DeKalb Medical Center (see directions below)
2701 North Decatur Road
Decatur, Georgia 30033

Speakers: Dr. Richard Clatch, Highland Park Hospital, Chicago, IL

From Steven.Gore at jhu.edu Mon Apr 24 07:21:49 2000
From: Steven.Gore at jhu.edu (Steven D. Gore)
Date: Mon, 24 Apr 2000 08:21:49 -0400
Subject: p21
References: <200004190546_MC2-A1C0-BF8D@compuserve.com>
Message-ID: <39043C5D.6FF90710@jhu.edu>


We validated and published a flow assay for p21 WAF1/CIP1 in Leukemia
13: 1243-53, 1999.

Steven Gore
Johns Hopkins Oncology Center



From Michael_Ormerod at compuserve.com Sun Apr 23 06:18:09 2000
From: Michael_Ormerod at compuserve.com (Michael Ormerod)
Date: Sun, 23 Apr 2000 07:18:09 -0400
Subject: Seeking Shareware/Freeware for Cell Cycle Analysis
Message-ID: <200004230718_MC2-A22D-AF12@compuserve.com>


You will find a free program for cell cycle analysis at
http://www.uwcm.ac.uk/uwcm/hg/hoy/software.html.

Michael Ormerod
34 Wray Park Road
Reigate RH2 ODE
Telephone: +44 (0)1737 241726
PLEASE NOTE NEW FAX & MOBILE NUMBERS
FAX: +44 (0)1737 226736
Mobile telephone: 07802 293242
Web site: http://ourworld.compuserve.com/homepages/Michael_Ormerod


From oamfilho at cpqrr.fiocruz.br Mon Apr 24 07:35:05 2000
From: oamfilho at cpqrr.fiocruz.br (Olindo Assis)
Date: Mon, 24 Apr 2000 09:35:05 -0300
Subject: Biotin-streptavidin staining
References: <38FCF61D.9C8BB9EF@garnet.tc.umn.edu>
Message-ID: <000601bfade9$86f74340$380983c8@cpqrr.fiocruz.br>


Dear Kevin, I think that if we do not wash the biotinylated conjugate before
adding the streptavidin (SA), we probably need more SA since the excess of
biotin-conjugate can remove a lot of unbound SA and your system will become
more expensive. Moreover, in cases where you have a little binding of your
biotin-conjugate to the target, the competition with the unbound ones could
generate false negative results.
Best regards,
Olindo





From scibox at online.nsk.su Sun Apr 23 12:08:13 2000
From: scibox at online.nsk.su (Pit Tarasov)
Date: Sun, 23 Apr 2000 23:08:13 +0600
Subject: can't contact Ray Lannigan
Message-ID: <10964.000423@online.nsk.su>


Hello,

I can't contact Ray Lannigan via e-mail
RLannigan at aol.com
Hope Ray is still reading this mail list
or someone else could help.

Sorry for disturbance,
Pit Tarasov
mailto:scibox at online.nsk.su




From DARZYNK at nymc.edu Sat Apr 22 10:17:20 2000
From: DARZYNK at nymc.edu (DARZYNKIEWICZ ZBIGNIEW)
Date: Sat, 22 Apr 2000 11:17:20 -0400
Subject: apoptosis and monocytes
Message-ID: <8C3E01568EC0D311AF0600508B0CC32B020F19EE@MAIL>




Maciej Simm wrote:
>
> One of the people I'm working with right now is studying apoptosis in
> periteneal macrophages using a kit with annexin V monoclonal FITC ab
> and propidium iodine.
>
> The cells are autofluorescent. The "unstained" tube has signal in up
> to 2nd log.
>



Monocytes/macrophages may often be positive (by flow cytometry) for several
apoptosis markers for the reason that they may have ingested apoptotic
bodies detached from genuine apoptotic cells. This is particularly evident
e.g. during aggressive chemotherapy of leukemias when many leukemic and
perhaps normal lymphocytes die by apoptosis. During ingestion of apoptotic
bodies most likely the plasma membrane of the latter fuses with the
macrophages' plasma membrane which leads to exposure of phosphatidylserine
on the surface and annexin V positivity of macrophages. The ingested
apoptotic bodies have fragments of chromatin and thus multiplicity of DNA
strand breaks, which makes them also positive in the TUNEL assay (e.g.see
Bedner et al., Cytometry, 35: 181-195,1999). I am guessing that such
monocytes may also be positive for for other markers e.g. these that detect
activated caspases, cleavage of PARP, etc. The only way to identify such
"false positive" cells is microscopy or Laser Scanning Cytometry, which
allows one to relocate them and examine whether they are loaded with
apoptotic bodies.
Zbigniew Darzynkiewicz


From tsuru at aichi-med-u.ac.jp Mon Apr 24 04:30:16 2000
From: tsuru at aichi-med-u.ac.jp (Masahito Tsurusawa)
Date: Mon, 24 Apr 2000 18:30:16 +0900
Subject: BMEC-1 cell lines
Message-ID: <39041428.50C17495@aichi-med-u.ac.jp>

Hi,

I am looking for the human
bone marrow microvascular
endothelial cell lines

to study the interaction of
leukemia cells and marrow
endothelial cells.

I found only one cell line
"BMEC-1"which is originated by
Candal et al.( Microvas res
52:221,1996)

by serching the medical
jouranls.

If anyone use BMEC-1or other
BM endothelial cell lines ,
tell me how can I get these
cells .

Thank you

Masahito Tsurusawa MD
associate professor of
pediatrics,
Aichi-medical University
Japan.

e-mail:
tsuru at aichi-med-u.ac.jp
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From donnenbergad at MSX.UPMC.EDU Fri Apr 21 16:41:46 2000
From: donnenbergad at MSX.UPMC.EDU (Donnenberg, Albert)
Date: Fri, 21 Apr 2000 17:41:46 -0400
Subject: EXPOv2 - EXPO32
Message-ID: <F2861F0327DCD111883F00805FA7FD5506BD04A1@1upmc-msx3.isdbu.upmc.edu>


Expo32 has just recently been released and we have not had much time with it
yet but so far, so good. It has many new features and has (hopefully)
resolved some of the problems in Version 2. Our early take is that it is
greatly improved.

There is still much to be said for the System II software. Despite the now
clunky DOS OS, they got many things right (especially regarding workflow for
a clinical lab or research lab that runs the same panels repetitively).

Albert D. Donnenberg, Ph.D.

Phone: (412) 624-9596
Fax: (412) 624-9624



-----Original Message-----
From: Tom Mc Closkey [mailto:thomasm at nshs.edu]
Sent: Wednesday, April 19, 2000 1:04 PM
To: cyto-inbox
Subject: Re: EXPOv2 - EXPO32





Marta.Borg at astrazeneca.com wrote:
>
> My question is, how can I know that EXPOv32 is better than EXPOv2 and why
> should I pay once more when I newer got what I paid for from the
beginning,
> an adequate software? (And I made the mistake to by 12 copies!)
> Is there anyone out there who has the same problem?
>
> Please all EXPO users, speak up!
>

Last summer, wanting to upgrade our DOS based computer on the Elite,
we
purchased a new EXPO workstation. We are also very disappointed with
this software [ver 2] as it is a step back in many regards from the DOS
based Elite software. When we communicated the deficiencies of this
software to Coulter we were told that a new version was being
developed. Still waiting. . .

Tom

****************************************************************************
*
Thomas W. Mc Closkey, Ph. D., Director of Flow Cytometry
North Shore University Hospital, New York University School of Medicine
Boas Marks Biomedical Research Center, 350 Community Drive
Manhasset, Long Island, New York 11030
ph: 516-562-4844 [office], 516-562-1135/4641 [lab] fax: 516-562-2866
****************************************************************************
*


From scibox at online.nsk.su Sun Apr 23 12:01:00 2000
From: scibox at online.nsk.su (Pit Tarasov)
Date: Sun, 23 Apr 2000 23:01:00 +0600
Subject: Cytometer wanted or parts
Message-ID: <5959.000423@online.nsk.su>


Dear Flowers,

We are located in Russia/Novosibirsk and looking
for cytometers (may be not working) or parts
of cytometer for give away or for cost close to
shipping cost.

We plan to use it on the experimental Scanning
Flow Cytometer unit, (principally new approach
to the light scattering)
http://www.kinetics.nsc.ru/llpc/cyto/.

All responses will be taken into consideration
with gratitude.

----------------------------------------------------
I must excuse for not responding to all of kind
suggestion in reply to my posting into the Cytometry
Mail List few months ago.
At that time we did not receive expected financial
support (now we did) and were unable to cover the
shipping cost of the cytometer.
----------------------------------------------------

Thank you,
Pit Tarasov
mailto:scibox at online.nsk.su

--------------------------------------------
you may contact my boss Valeri Maltsev
directly at:

Flow Cytometry Center
E-mail: MALTSEV at NS.KINETICS.NSC.RU
http://www.kinetics.nsc.ru/llpc/maltsev/

Institute of Chemical Kinetics & Combustion
Institutskaya 3 Novosibirsk 630090, Russian Federation.
Voice: +7-3832-333240(w), -366737(h)
FAX: +7-3832-342350 (to V.Maltsev)




From alagoo at pathology.umsmed.edu Fri Apr 21 12:26:53 2000
From: alagoo at pathology.umsmed.edu (Anand Lagoo)
Date: Fri, 21 Apr 2000 11:26:53 -0600
Subject: CD11b expression on Lymphocytes
Message-ID: <s9003b17.064@gwia.umsmed.edu>


CD18 combines with CD11a (to form LFA-1, expressed mainly on lymphocytes), or CD11b
(to form MAC-1, expressed mainly on macrophage with low expression on lymphocytes)
or with CD11c (to form a iC3b receptor, expressed mainly on macrophage). The last two
are decreased in congenital "leukocyte adhesion deficience". Therefore, low expression
of CD11b in total leukocyte will be of concern, but on lymphocytes CD11b is normally
expected to be much lower than CD18 (where it is paired mostly with CD11a).
For details about this and other CDs visit:

http://www.ncbi.nlm.nih.gov/prow/

Hope that helps.


Anand Shreeram Lagoo, MD, PhD
Associate Professor
Department of Pathology
University of Mississippi Medical Center
2500 N State Street
Jackson, MS 39216
(601) 984-1530; (601)984-1531 fax; (601)478-0344 pager

>>> Maciej Simm <simmmmer at yahoo.com> 04/19/00 03:37PM >>>

Greetings once again,

We have recently received a blood specimen and were asked to stain
for CD18/11b ( I love it how doctors don't explain the condition of
the patient ahead of time, so we know what to look for )

We found, that when compared to our NC's (N=300+ for this "tube" and
growing) this patient had less +/+ lymphoctes (~30% vs. 12%). The
expression of CD18 was not reduced - all of his leukocytes had >97%.

A question came up: what is the significance of CD18+/CD11b-
lymphocytes? what is the significance of a decrease in the +/+
population?

We do not routinely do CD11a or CD11c - are these mutually exclusive?
I.e. it's either a, b or c but never any of them together?

And finally, is the ligand for CD11b soluble and may have interfered
with our staning?

This staining was done in whole blood (which I just found is a
no-no).

I would appreciate if anyone could share any thoughts on this topic.

Maciej

__________________________________________________
Do You Yahoo!?
Send online invitations with Yahoo! Invites.
http://invites.yahoo.com



From pxpetit at cochin.inserm.fr Sat Apr 22 05:05:44 2000
From: pxpetit at cochin.inserm.fr (Patricex Petit)
Date: Sat, 22 Apr 2000 12:05:44 +0200
Subject: JC-1 positive test
Message-ID: <l03130305b527224b6d43@[193.52.75.146]>


ANSWER TO

X-Sender: butterfield.15 at pop.service.ohio-state.edu
If anyone has used the mitochondrial membrane probes such as JC-1 or
MitoTracker can you tell me what works as a positive control?

Thanks.

Dear Butterfield,

At first, take care to use low probe conecntration, and this is the case
for all the probes.
inthe nM range will be the best.
The control is often to collapse to mitochondria membrane potential with
decoupling agents
which could be FCCP or mClCCP (sigma). You may realize a titration from 1
micromolar up to sometime 50 micromolar depending of the cell type and of
the medium used because the FSC in the medium or the others proteins will
trap part of the decoupling compounds.

See first Rottenberg et al. (1998) and our proper publication taking into
account that the amount of probe has to be reduced as much as possible up
to 1nM.

1.Rottenberg, H. & Wu, S. Quantitative assay by flow cytometry of the
mitochondrial membrane potential in intact cells. Biochim. Biophys. Acta
1404, 393-404. (1998).
2.Petit, P.X., O'Connor, J.E., Grunwald, D. & Brown, S.C. Analysis of the
membrane potential of rat- and mouse-liver mitochondria by flow cytometry
and possible applications. Eur. J. Biochem. 220, 389-397 (1990).
3.Zamzami, N. et al. Reduction in mitochondrial potential constitutes an
early irreversible step of programmed lymphocyte death in vivo. J. Exp.
Med. 181, 1661-1672 (1995).
4.Petit, P.X. et al. Alterations of mitochondrial structure and function
are early events of dexamethasone-induced thymocyte apoptosis. J. Cell.
Biol. 130, 157-167 (1995).

Sincerely yours

Patrice


?????????????????? \ | /
?????????????????? (o o) \ | /
_______________oOo_____oOo_____oOo_? (o o)__oOo________________

Dr. Petit Patrice X.
Institut Cochin de G?n?tique Mol?culaire
INSERM U129 - CHU Cochin Port-Royal
24, rue du Faubourg Saint-Jacques
F-75014 Paris, France.

Tel: 33 01 44 41 24 11
Fax: 33 01 44 41 24 21
E-mail: pxpetit at icgm.cochin.inserm.fr




From mandy_cromwell at hms.harvard.edu Fri Apr 21 17:51:01 2000
From: mandy_cromwell at hms.harvard.edu (mandy cromwell)
Date: Fri, 21 Apr 2000 15:51:01 -0700
Subject: CD11b expression on Lymphocytes
In-Reply-To: <20000419213735.22592.qmail@web2003.mail.yahoo.com>
References: <20000419213735.22592.qmail@web2003.mail.yahoo.com>
Message-ID: <v04210100b526867fd86f@[128.103.82.205]>


Maciej,

While I can't offer a clinical perspective on your finding and its
significance, here is some background on CD11b/CD18:

CD18 can combine with the CD11a,b or c subunits to form to form the
integrins LFA-1, Mac-1 and p150,95. The CD11b/CD18 +/+ cells are
expressing Mac-1, but can also have the other integrins. The
CD18+/11b- cells are negative for Mac-1, but are expressing one or
more of the other CD18-utilizing integrins.

One of the ligands for Mac-1 is iC3b, a complement component which is
of course soluble; other soluble ligands are fibrinogen and factor X.
These could be one reason why staining whole blood is contraindicated.

Do you know whether the patient has a leukocyte adhesion defect? I
believe this is more likely to be seen with loss of CD18, where all 3
integrins are missing. CD11b is also highly expressed on CD8+
effector T lymphocytes (Hamann, D, J.Exp. Med., 1997); and you are
only seeing a decrease, not an absence of CD11b+ cells.

I'm sure there are other readers who can give you more in-depth info
on this, but its a start. For a reference try: Larson, RS and
Springer,TA (1990) Immunol. Rev. 114, 181-217.

good luck,

Mandy

>Greetings once again,
>
>We have recently received a blood specimen and were asked to stain
>for CD18/11b ( I love it how doctors don't explain the condition of
>the patient ahead of time, so we know what to look for )
>
>We found, that when compared to our NC's (N=300+ for this "tube" and
>growing) this patient had less +/+ lymphoctes (~30% vs. 12%). The
>expression of CD18 was not reduced - all of his leukocytes had >97%.
>
>A question came up: what is the significance of CD18+/CD11b-
>lymphocytes? what is the significance of a decrease in the +/+
>population?
>
>We do not routinely do CD11a or CD11c - are these mutually exclusive?
>I.e. it's either a, b or c but never any of them together?
>
>And finally, is the ligand for CD11b soluble and may have interfered
>with our staning?
>
>This staining was done in whole blood (which I just found is a
>no-no).
>
>I would appreciate if anyone could share any thoughts on this topic.
>
>Maciej
>
>__________________________________________________
>Do You Yahoo!?
>Send online invitations with Yahoo! Invites.
>http://invites.yahoo.com



From royal011 at visto.com Sun Apr 23 10:53:19 2000
From: royal011 at visto.com (Bratislav Janjic)
Date: Sun, 23 Apr 2000 08:53:19 -0700
Subject: [No Subject]
Message-ID: <39005FA000015789@smtp.visto.com> (added by administrator@visto.com)

Hi,

I work on FACScan cytometer and I have a problem that is shown in attachment "Problem": dendritic cell subpopulation that I analyze is always on the right side so I can not see all subpopulation in FSC/SSC dotplot diagram. So, is there any possibility to see all subpopulation, or that is the characteristic of my device that I can not change.
My next question is: if I analyze those cells in FL1/FL2, what's happen with cells that are also labeled with proper Abs, but I didn't see them on FSC/SSC dotplot (because they are too much on the right side of FSC) to gate them.

By the way,
My name is Bratislav Janjic and I am B.Sc. of Biology. I work in Department of Pathology, University of Pittsburgh, Pennsylvania, USA and e-mail address is: royal011 at visto.com
I'm still not member of your society, but I hope that I can get application form to become it.
Thank's in advance.

Bratislav Janjic



__________________________________________________________________________
Visit http://www.visto.com/info, your free web-based communications center.
Visto.com. Life on the Dot.
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From mroy at cmgm.stanford.edu Mon Apr 24 07:31:59 2000
From: mroy at cmgm.stanford.edu (Meenakshi Roy)
Date: Mon, 24 Apr 2000 05:31:59 -0700
Subject: markers for non-recirculating memory B cells?
Message-ID: <3.0.2.32.20000424053159.00704d68@cmgm.stanford.edu>



Dear flow-ers,
I am told there are two populations of memory B cells--one
non-recirculating (L-selectin -ve and CD11b +ve) and one recirculating
(L-selectin +ve and CD11b -ve). I have found some references about this,
but they are in sheep.
Can anyone enlighten me about this?

Meenakshi Roy


From simmmmer at yahoo.com Mon Apr 24 15:42:23 2000
From: simmmmer at yahoo.com (Maciej Simm)
Date: Mon, 24 Apr 2000 13:42:23 -0700 (PDT)
Subject: quenching
Message-ID: <20000424204223.222.qmail@web2003.mail.yahoo.com>


in my humorous approach, I'd call it

"Quenching is as quenching does"

and then show a tissue slide stained with a peroxidase conjugated
antibody unquenched :)

Then go into the discussion of what quenching is - eliminating the
activity of some enzyme by saturating it with its substrate.

Then if anyone asks why it needs to be done, show them that slide
again :)



Better yet,

"This is your tissue slide. This is your tissue slide after
quenching. Any questions?"

__________________________________________________
Do You Yahoo!?
Send online invitations with Yahoo! Invites.
http://invites.yahoo.com


From Sciex at AOL.COM Mon Apr 24 12:12:13 2000
From: Sciex at AOL.COM (Sciex@AOL.COM)
Date: Mon, 24 Apr 2000 13:12:13 EDT
Subject: mitochondrial probes
Message-ID: <6b.372285d.2635da6d@aol.com>


Exalpha offers a monoclonal antibody for mitochondrial staining (cat A115M),
useful for western blots or ih.
ref.
The 2G2 antibody recognizes an acidic 110-kDa human mitochondrial protein.
Paulin-Levasseur M; Chen G; Larivi`ere C. Histochem J, 30(9):617-25 1998 Sep

You can reach Exalpha at; 800. 395. 1137 or 617. 445. 6463
email: info at exalpha.com

Regards,
John


From Laurie_Gilmour at BDIS.Com Mon Apr 24 15:52:23 2000
From: Laurie_Gilmour at BDIS.Com (Laurie_Gilmour@BDIS.Com)
Date: Mon, 24 Apr 2000 13:52:23 -0700
Subject: cloning question
Message-ID: <882568CB.00736263.00@bdis.com>



I am forwarding a message from a colleague. Please reply directly to her:
mcook at iconixpharm.com
---------------------- Forwarded by Laurie Gilmour/SANJ/BDX on 04/24/2000 01:51
PM ---------------------------


Melinda Cook <mcook at iconixpharm.com> on 04/18/2000 04:32:28 PM

To: cyto-inbox
cc:
Subject: cloning question




I am having problems with me cloning efficiency and I wanted to find out
what % other
people are getting with the Vantage SE. We are finding about 30-35% of our
wells from a
96 well plate have colonies after three weeks. If people are getting >35% I
would be
interested in what you are doing to achieve that %.
Melinda

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From larry.e.kahn at stl.monsanto.com Mon Apr 24 15:45:05 2000
From: larry.e.kahn at stl.monsanto.com (KAHN, LARRY E [PHR/1005])
Date: Mon, 24 Apr 2000 15:45:05 -0500
Subject: Position Available
Message-ID: <82136906C193D311976500A0C9EAD0484B85AD@ems1000-03.monsanto.com>


Searle:

It's the people, the complex challenges, the stimulating environment.
The opportunity to work independently while contributing to a team.
The opportunity to make a difference - to leave your mark.
It's about food, health, hope and an exciting future as we merge with
Pharmacia/Upjohn to form the new Pharmacia Corporation.

Research
Flow Cytometry Specialist

A position is available for a flow cytometry specialist within Searle
Discovery Research to join multidisciplinary teams focused on the discovery
of therapeutics related to oncology. We seek an individual who is
self-motivated, innovative, and has strong communication skills. The
primary responsibilities are to provide analytical support and to help
integrate cytometry approaches into current and future research efforts. We
are seeking an individual with 2-5 years of "hand-on experience" on both
analyzers and cell sorters. Multi-laser, high speed sorting, assay
development and training experience is a plus. The successful candidate
should also have a strong background in biological sciences to contribute to
experimental design.

We offer a competitive salary and benefits package including tuition
reimbursement and 401(k). If you meet these basic qualifications, and think
that you would thrive in our special environment, please submit your resume
including three letters of recommendation to: Staffing, 700 Chesterfield
Parkway, Mail zone: BB4D, Chesterfield, MO 63198. Attn. Position #00-0436
EEO/AA Employer M/F/D/V.

Please visit our website at www.searle-ican.com <http://www.searle-ican.com>




From mtropea at nih.gov Tue Apr 25 04:48:06 2000
From: mtropea at nih.gov (Margaret Tropea)
Date: Tue, 25 Apr 2000 10:48:06 +0100
Subject: Receptors
In-Reply-To: <000e01bfaa34$33012e80$380983c8@cpqrr.fiocruz.br>
References: <fc.004c51fa00448c5a004c51fa00448c5a.448c81@pdl.com>
Message-ID: <v04011704b52b196791a9@[128.231.81.17]>

Flow Cytometry Stds. also has a system for determining ABC with beads and
software. They can be reached @ 800-227-8143.
Margaret


>I know that BD has a system called quantiBright (or QuantiBrite) that can be
>used for these purpose.
>Also STAGO has a similar product designed to analysis of receptors on
>platelets.
>
>Good luck,
>
>Olindo
"The opinions stated above are the authors own, and should not be construed as
U.S. Government endorsement of a specific vendor, nor used in any publication
to indicate endorsement by the U. S. government or any of its agencies."

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
Margaret M. Tropea
Critical Care Medicine Department
National Institutes of Health
(ph) 301-496-7752
(fax) 301-480-3389
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
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From Carol.W.Johnson at ap.pnu.com Tue Apr 25 09:53:29 2000
From: Carol.W.Johnson at ap.pnu.com (Carol.W.Johnson@ap.pnu.com)
Date: Tue, 25 Apr 2000 10:53:29 -0400
Subject: apoptosis and monocytes
Message-ID: <852568CC.0051CE1B.00@ap.pnu.com>




Has anyone actually looked at the cells with a fluorescent microscope?

We found a similar signal for rat bone marrow cells, but instead of
autofluorescence, the cells had speckled surface staining
with Annexin V.

I agree with Dr Darzynkewicz that microscopy could be of value when an unusual
Flow Cytometry signal is present.

Carol W Johnson DVM PhD
Pharmacia Corp.








DARZYNKIEWICZ ZBIGNIEW <DARZYNK at nymc.edu> on 04/22/2000 11:17:20 AM

To: cyto-inbox
cc: (bcc: Carol W Johnson/USKZO/PNU)
Subject: apoptosis and monocytes







Maciej Simm wrote:
>
> One of the people I'm working with right now is studying apoptosis in
> periteneal macrophages using a kit with annexin V monoclonal FITC ab
> and propidium iodine.
>
> The cells are autofluorescent. The "unstained" tube has signal in up
> to 2nd log.
>



Monocytes/macrophages may often be positive (by flow cytometry) for several
apoptosis markers for the reason that they may have ingested apoptotic
bodies detached from genuine apoptotic cells. This is particularly evident
e.g. during aggressive chemotherapy of leukemias when many leukemic and
perhaps normal lymphocytes die by apoptosis. During ingestion of apoptotic
bodies most likely the plasma membrane of the latter fuses with the
macrophages' plasma membrane which leads to exposure of phosphatidylserine
on the surface and annexin V positivity of macrophages. The ingested
apoptotic bodies have fragments of chromatin and thus multiplicity of DNA
strand breaks, which makes them also positive in the TUNEL assay (e.g.see
Bedner et al., Cytometry, 35: 181-195,1999). I am guessing that such
monocytes may also be positive for for other markers e.g. these that detect
activated caspases, cleavage of PARP, etc. The only way to identify such
"false positive" cells is microscopy or Laser Scanning Cytometry, which
allows one to relocate them and examine whether they are loaded with
apoptotic bodies.
Zbigniew Darzynkiewicz






From lkrueg at ix.netcom.com Tue Apr 25 06:45:37 2000
From: lkrueg at ix.netcom.com (lori krueger)
Date: Tue, 25 Apr 2000 07:45:37 -0400
Subject: Platelet Research Position Available
Message-ID: <003d01bfaeab$c6e06aa0$dea4fea9@8skha>

I am posting this message on behalf of Dr. Alan D. Michelson, M.D.
Please respond directly to him at Michelson at platelets.org

Thanks,

Lori Krueger
krueger at platelets.org

____________________________________________________________

PLATELET RESEARCH POSITION AVAILABLE


The Center for Platelet Function Studies at the University of Massachusetts
Medical Center in Worcester (45 miles west of downtown Boston) is looking
to hire an additional research associate.


The majority of the work will involve platelet flow cytometry.


The type of work we currently do is outlined in the review article:
Michelson AD. Flow cytometry: a clinical test of platelet function. Blood
87:4925-4936, 1996.


The research associate will actively participate in, and be a co-author of,
an interesting variety of state-of-the-art research studies of platelet
function.

The ideal candidate will have flexible hours (dependent on when research study samples arrive)to include some evenings and weekends but the total number of hours per week will not exceed 40.


The minimum qualification is a bachelor's degree or medical laboratory
technology diploma plus 2 years of research experience. Platelet research is not essential.


If you are potentially interested, please contact Dr. Alan Michelson for
further details. Telephone: (508) 856-0056, FAX: (508) 856-4282.

E-mail: Michelson at platelets.org

Alan D. Michelson, M.D.

Director, Center for Platelet Function Studies
Professor of Pediatrics and Pathology
University of Massachusetts Medical Center
Room S5-846
55 Lake Avenue North
Worcester, MA 01655


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From maris_handley at dfci.harvard.edu Tue Apr 25 19:27:49 2000
From: maris_handley at dfci.harvard.edu (Maris Handley)
Date: 26 Apr 00 00:27:49 +0000
Subject: Sources for murine GM-CSF
Message-ID: <200004260427.XAA27481@flowcyt.cyto.purdue.edu>


Hello Everyone,

A researcher here has asked me to post this; you can reply to him directly, or I will
pass along any information sent to me.

We are looking for a source for anti-mouse CD116 GM-CSF R alpha chain
and CDw131 IL-3, IL-S, GM-CSF B common chain.

Thanks (as always) for your help.
Maris

reply to: brian_wilson at dfci.harvard.edu





Maris Handley
Dana-Farber Cancer Institute
J415
44 Binney St.
Boston, MA 02115
maris_handley at dfci.harvard.edu




From koratich at sri.org Tue Apr 25 12:25:40 2000
From: koratich at sri.org (Koratich, Mike)
Date: Tue, 25 Apr 2000 12:25:40 -0500
Subject: Job Openings
Message-ID: <346BEE1396CFD311B56B0008C7B17800454607@srint3.sri.org>



Research Associate, Cell Biology and Immunology Group

SERQUEST
A division of Southern Research Institute


Serquest, a Division of Southern Research Institute, conducts comprehensive
pre-clinical drug development and testing as well as basic research in drug
design and synthesis, pharmaceutical formulations, toxicology, virology,
microbiology, and pharmacology. The Cell Biology and Immunology group within
the Cancer Therapeutics Department of Serquest is currently seeking
candidates for the position of Research Associate. This position is
responsible for performing a broad range of biological tests and procedures
using standard scientific techniques and methods; and recording, analyzing,
and reporting results.

PRIMARY ACTIVITIES

* Resolve problems requiring the application of standard procedures
and techniques.
* Conduct experimental procedures.
* Collect and analyze data for review by management, utilizing
sophisticated spreadsheets and statistical analysis software.
* Exercise judgement in conducting experimental procedures,
recognizing deviations from expected results and postulating sources of
deviations.
* Maintain knowledge of scientific discipline through familiarity with
current scientific literature.
* Arrive at preliminary interpretations.
* Observe appropriate safety and study requirements by reading,
understanding, and following Standard Operating Procedures (SOP), Good
Laboratory Practices (GLP) requirements, and study protocols.
* Observe appropriate safety and health practices including Personal
Protective Equipment (PPE) and Safe Laboratory Practices (SLP).
* Perform other duties as may be required by supervisor.
* Resolve problems where analysis of data requires creative insight.
* Train and check the work of less experienced personnel and new
technicians.
* Perform preliminary calculations and summarize data.

MINIMUM REQUIREMENTS AND QUALIFICATIONS

* BS degree in a relevant scientific discipline.
* At least one year of directly related laboratory experience.

DESIRED SKILLS AND QUALIFICATIONS

* Knowledge of and ability to use the fundamental concepts, practices,
and procedures of cell biology, molecular biology and immunology.
* Knowledge of cell culture techniques.
* Knowledge of computer software used for assays.
* Knowledge of radiation handling and protection.


Cell/Molecular Biologist/Immunologist
SERQUEST
A Division of Southern Research Institute
Birmingham, Alabama

Serquest, a Division of Southern Research Institute, conducts comprehensive
pre-clinical drug development and testing as well as basic research in drug
design and synthesis, pharmaceutical formulations, toxicology, virology,
microbiology, and pharmacology. The Cell Biology and Immunology group within
the Cancer Therapeutics Department of Serquest is currently seeking
candidates for the position of Research Scientist. The successful candidate
will interface with clients and potential clients, design projects and
individual studies as well as oversee the daily operation of a cellular and
molecular biology laboratory including managerial responsibilities. In
addition, the successful candidate will generate cost estimates and
proposals for studies, oversee the implementation of the studies/projects,
write interim and final reports, and assure the completion of tasks and
projects within constraints of time, quality, and cost. Individuals
applying for this position should be capable of directing other scientists,
enthusiastic about constantly improving the functions of the laboratory and
able to work effectively with a minimum of supervision. The position
requires an M.S. with extensive experience or Ph.D. in Cell Biology,
Molecular Biology, Immunology, or other biological science or cancer
research related field. Experience in a commercial research setting is an
asset but not required.

Southern Research Institute, a non-profit research organization, employs
over 500 professional, technical and support personnel to conduct research
with the NIH, other government organizations and commercial clients. The
Institute is located in Birmingham, Alabama, a cosmopolitan city of over a
million people. Birmingham has a high standard of living afforded by a
moderate climate, beautiful wooded terrain, outstanding residential
communities and schools, world-class healthcare, below average cost of
living and a vibrant arts and entertainment scene.

Serquest offers an excellent compensation package that includes a
competitive salary and comprehensive benefits package. Inquiries may be
directed to Dr. Alex Aller, Manager, Cell Biology and Immunology Group at
(205) 581-2731 or aller at serquest.com. Please send your resume to Sheryl H.
Brown, Human Resources, Dept. 580, P.O. Box 55305, Birmingham, AL
35255-5305. Phone: (205) 581-2512. Fax: (205) 581-2880. E-mail:
s.r.brown at sri.org EOE/AA



From rgniadecki at hotmail.com Wed Apr 26 03:26:13 2000
From: rgniadecki at hotmail.com (robert gniadecki)
Date: Wed, 26 Apr 2000 01:26:13 PDT
Subject: monocyte marker
Message-ID: <20000426082613.47837.qmail@hotmail.com>


Hello!
Could anybody suggest a single, good and reproducible antibody for
unequivocal identification of monocytes/macrophages in flow in peripheral
blood? (I cannot use forward/side scatter in my equipment - I have LSC).
Thanks,
Robert Gniadecki, MD
Dermatology, University of Copenhagen
Denmark
rgniadecki at hotmail.com

________________________________________________________________________
Get Your Private, Free E-mail from MSN Hotmail at http://www.hotmail.com



From hakari at niaid.nih.gov Wed Apr 26 09:15:48 2000
From: hakari at niaid.nih.gov (Hirofumi Akari)
Date: Wed, 26 Apr 2000 10:15:48 -0400
Subject: CD4/CD8 coexpression in monkey
Message-ID: <v04010103b52caa94e8f4@niaid.nih.gov>


Dear everyone,
We have been interested in DP T cells in cynomolgus monkeys and found that
the DP T cell subpopulation is CD8alpha/alpha, CD28-, CD29hi, CD69-, CD49dhi,
and CD80+. Uniquely, the subpopulation increases with age (<9 years old;
less than 5%, >10 years old; more than 10% in average).

Reference:
Int. Immunol 9, 591, 1997
Dev. Comp. Immunol. 22, 239, 1998

Hirofumi Akari, DVM & Ph.D.
NIAID, NIH


From hakari at niaid.nih.gov Tue Apr 25 11:08:13 2000
From: hakari at niaid.nih.gov (Hirofumi Akari)
Date: Tue, 25 Apr 2000 12:08:13 -0400
Subject: CD4/CD8 coexpression in pb
Message-ID: <v04010103b52b736d74b0@niaid.nih.gov>


Dear Andrea,
We have been interested in DP T cells in cynomolgus monkeys and found that
the DP T cell subpopulation is CD8alpha/alpha, CD28-, CD29hi, CD69-, CD49dhi,
and CD80+. Uniquely, the subpopulation increases with age (<9 years old;
less than 5%, >10 years old; more than 10% in average).

Reference:
Int. Immunol 9, 591, 1997
Dev. Comp. Immunol. 22, 239, 1998

Hirofumi Akari, DVM & Ph.D.
NIAID, NIH
*****************************************************
Hirofumi Akari, D.V.M. & Ph. D.
Viral Biochemistry Section, Laboratory of Molecular Microbiology
National Institute of Allergy and Infectious Diseases
National Institutes of Health, 4/312, Center Drive MSC,
Bethesda, MD 20892
phone: 301-496-3132, fax: 301-402-0226
*****************************************************


From granzyme at merle.acns.nwu.edu Tue Apr 25 17:05:01 2000
From: granzyme at merle.acns.nwu.edu (Christopher Froelich, M.D.)
Date: Tue, 25 Apr 2000 17:05:01 -0500
Subject: Research Position in Cytotoxic Cell Granule Mediated Apoptosis
Message-ID: <v04220800b52bc0a1ed68@[192.168.1.1]>


Dear Flow Community:

My lab is looking for a research associate (BS/MS with flow and
biochemistry expertise) who would be assigned to projects designed to
understand how perforin delivers granule proteases to target cells.
We are situated in Evanston, IL - north of Chicago - on the lake
front. Individual would join two post-docs and another technician.
A specific detailed job description is available on request. ENH is
an equal opportunity employer.

Thanks

Chris Froelich
Christopher J. Froelich, M.D.
Associate Professor
Evanston Northwestern Research Institute
ENH Women's Hospital, Room B624
2650 Ridge Avenue
Evanston, IL 60201-1797

Voice (off) 847.570.2438
Voice (lab) 847.570.2646
Fax 847.570.1253
e-mail granzyme at merle.acns.nwu.edu

http://x.biochem.nwu.edu/ibis/research/froelich.html



From anardin at idm-biotech.com Wed Apr 26 03:47:07 2000
From: anardin at idm-biotech.com (Alessandra Nardin)
Date: Wed, 26 Apr 2000 10:47:07 +0200
Subject: DR3+ cell line
Message-ID: <200004260847.KAA20588@william.bhdc.jussieu.fr>



Hi all,

does anyone of you know if and where it is available a DR3+ cell line? We
are trying to see if we can set up a HLA typing at home and I am worring
about controls...

Thanks a lot for any answer.

alessandra







--

Alessandra Nardin

LUTI-IDM
Institut Biom?dical des Cordeliers
15, rue de l'Ecole de M?decine
75006 Paris

tel. +33 (0)1 53 10 17 75
fax +33 (0)1 53 10 17 80




From cabrera.ej at pg.com Tue Apr 25 13:06:17 2000
From: cabrera.ej at pg.com (Ed Cabrera-EJ)
Date: Tue, 25 Apr 2000 14:06:17 -0400
Subject: sICAM-1
Message-ID: <852568CC.0063742B.00@bdc-notes082.na.pg.com>


This is not directly related to cytometry, but perhaps someone in this
conference has some insight into this issue.
I am interested in measuring sICAM in swine plasma. There are kits available
for human sICAM. Does anyone know if there is a good crossreactivity between
human and swine sICAM. I have shown that neutrophils will adhere to HUVEC thru
a mechanism that is inhibited by swine antiCD18, so I assume that there is a
certain degree of crossreactivity since ICAM-1 is the main counterreceptor for
CD18/CD11b.

Any help will be appreciated.

Thanks

Ed Cabrera
Procter and Gamble Pharmaceuticals
Mason, OH




From stoutej at net2000ke.com Tue Apr 25 22:29:49 2000
From: stoutej at net2000ke.com (Jose A. Stoute)
Date: Wed, 26 Apr 2000 06:29:49 +0300
Subject: Primary Conjugated antibodies against CD35 and CD55
Message-ID: <390662AD.FEA9D116@net2000ke.com>


Dear group, I am trying to setup assays of direct fluorescent staining
of RBCs for CD35 and CD55. All the primary conjugated antibodies I have
tried for these antigens that are commercially available give a very dim
response, especially for CD35 which is known to have low expression on
RBCs. I have had good results using indirect fluorescence using a
secondary anti-mouse but this would preclude multicolor analysis, hence
we would like to switch to direct fluorescence. Any suggestions as to
which antibodies I could use or the approach?

Thank you in advance for your assistance.

Jose A. Stoute

--
Jose A. Stoute, MD
Unit 64109, Box 401
USAMRU-Kenya
APO AE 09831-4109
e-mail: stoutej at net2000ke.com
Nairobi Tel 254-2-729303, Fax 254-2-714592
Kisumu Tel 254-35-22942, Fax 254-35-22903
Electronic Fax Service: 1-630-214-2008, 1-917-463-0373, 1-917-477-6048





From waddi002 at tc.umn.edu Tue Apr 25 16:06:41 2000
From: waddi002 at tc.umn.edu (Kevin Waddick)
Date: Tue, 25 Apr 2000 16:06:41 -0500
Subject: EMA
Message-ID: <390608D3.7937260C@garnet.tc.umn.edu>


A week ago Howard Shapiro enlightened me (and maybe others) by
saying that, whereas propidium is (supposedly) membrane impermeant to
healthy cells, ethidium is membrane permeant and is pumped out of
healthy cells. Thus, propidium is a viability stain that operates by
passive dye exclusion and ethidium requires active participation on the
part of the healthy cell to appear negative for staining with this dye.
I am wondering if newly thawed cells have the ability to pump out
ethidium -- or, more specifically, ethidium monoazide (EMA) -- if they
are otherwise viable at that point. When I thaw cryopreserved cells I
get a clear-cut idea of their viability immediately afterward by trypan
blue exclusion (I realize that early apoptotic cells are trypan
negative) because it is a yes-or-no decision. When I stain with 5 ug/mL
EMA, leave the cells in the dark for 15 minutes and then expose them to
a nearby fluorescent light source for 15 minutes, I get a confusing
pattern when the cells are analyzed by flow cytometry after washing,
fixing, etc. Not even all of the cells obviously dead by FSC, SSC, and
hypodiploidy are positive for EMA staining. And the seemingly viable
cells have a wide range of EMA positivity, whereby many of them have
higher intensity than the lower intensity dead cells.
So, can EMA even be used right after cells have been thawed, or is
their permeability/dye pumping status totally messed up? I would like to
be able to quickly fix/permeabilize cryopreserved cells after thawing to
prevent further cell death and in preparation for intracellular staining
but be able to distinguish the cells that were dead prior to that point.

Also, is EMA unstable in solution at 4 C or RT, unlike plain old
ethidium bromide? The results I got soon after making a stock solution
of 0.5 mg/mL EMA were quite different from those 2 weeks later when the
stock had been sitting in the refrigerator.

Kevin G. Waddick, Ph.D.
Parker Hughes Institute
2657 Patton Road
St. Paul, MN 55113



From rgniadecki at hotmail.com Thu Apr 27 08:00:04 2000
From: rgniadecki at hotmail.com (robert gniadecki)
Date: Thu, 27 Apr 2000 06:00:04 PDT
Subject: Resume: monocyte marker
Message-ID: <20000427130004.63922.qmail@hotmail.com>


I would like to thank everybody who answered my question. Everybody agreed
that CD14 is the best macrophage/monocyte marker.
Thanks again,
Robert Gniadecki
Copenhagen
________________________________________________________________________
Get Your Private, Free E-mail from MSN Hotmail at http://www.hotmail.com



From dawes.sm at pg.com Wed Apr 26 15:42:44 2000
From: dawes.sm at pg.com (Susan Dawes-SM)
Date: Wed, 26 Apr 2000 16:42:44 -0400
Subject: dye laser manual
Message-ID: <852568CD.0071C73B.00@bdc-notes082.na.pg.com>


Hello All,

Cleaning out the cabinets, I found the manual for the 599 Dye Laser (Coherent)
from the Coulter 753 which left the lab many moons ago. Is anyone willing to
admit they still run the dye laser, and have interest in owning the manual?
Please contact me directly for acquisition. Thanks

Sue Dawes
dawes.SM at PG.com




From oamfilho at cpqrr.fiocruz.br Thu Apr 27 07:06:55 2000
From: oamfilho at cpqrr.fiocruz.br (Olindo Assis)
Date: Thu, 27 Apr 2000 09:06:55 -0300
Subject: monocyte marker
References: <20000426082613.47837.qmail@hotmail.com>
Message-ID: <000601bfb041$15c5a360$380983c8@cpqrr.fiocruz.br>


Hi Robert,

For Monocytes in PB you can use CD14/CD45 conbination that will allow you to
discriminate monocytes as CD14 high (bright) and CD45 medium (between
granulocytes and lymphocytes). This conbination is considered for many
scientist as the best way to gate the three majos leucocytes populations in
flow (known as leucogate).

I hope it helps,


Olindo



From b.windsor at mail.utexas.edu Thu Apr 27 11:03:13 2000
From: b.windsor at mail.utexas.edu (J.Brian Windsor)
Date: Thu, 27 Apr 2000 09:03:13 -0700
Subject: micelle or liposome sorting
Message-ID: <l03110700b52e1477bf46@[146.6.170.201]>


Does anyone have any information on sorting micelles, liposomes, or
liposome-like compartments? I would like to do in vitro
transcription/translation in some sort of compartment (about 1.5 micron
diameter) and directly measure properties using flow cytometry (NOT fuse
these liposomes to cells, then sort). I found a protocol for doing this
sort of transcription/translation in a water/mineral oil emulsion, but I'm
pretty sure that I can't run mineral oil through the cytometer.

Any ideas? Thanks-


Brian Windsor
Alan Lloyd Lab ICMB
The University of Texas at Austin
MBB 1.448B mail code: A4800
Austin, Texas 78713
(512) 471-3553
fax: (512) 232-3432
b.windsor at mail.utexas.edu




From faceburn at netzero.net Thu Apr 27 08:56:27 2000
From: faceburn at netzero.net (Jens Reindl)
Date: Thu, 27 Apr 2000 08:56:27 -0500
Subject: FACScan offer
Message-ID: <001001bfb050$693338a0$6e823604@net>

"Used FACScan for sale"
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From VFalco at Lifespan.org Wed Apr 26 14:49:46 2000
From: VFalco at Lifespan.org (Falco, Vincent)
Date: Wed, 26 Apr 2000 15:49:46 -0400
Subject: RESERACH ASSISTANT MOLECULAR CYTOGENETICS
Message-ID: <477561A36172D311BE7600508B139F85429918@neexch02.nemc.org>


Research Assistant II - Molecular Cytogenetics

The New England Medical Center, Boston, MA., is seeking a qualified
applicant with 1-2 years experience in molecular cytogenetics. Candidates
should be proficient in FISH, chromosome probe development, and production
techniques as PCR and probe labeling. Experience in light and fluorescent
microscopy, image analysis workstation and image capture tools. Secondary
experience in Flow Cytometry a plus.

The Division of Genetics Research is well funded conducting projects in
fetal and prenatal diagnostics, michro-chimerism, stem cell recovery and
culturing, and fetal-maternal cell trafficking.

Forward resume to VFalco at Lifespan.org or mail to:

New England Medical Center
Attention: Vincent Falco
750 Washington Street Box 394
Boston, MA. 02111



From Diane.M.Sharp at dupontpharma.com Wed Apr 26 14:13:31 2000
From: Diane.M.Sharp at dupontpharma.com (Diane M Sharp)
Date: Wed, 26 Apr 2000 15:13:31 -0400
Subject: BrdU/PI cell cycle analysis
Message-ID: <0221C39073FDB006M/@MHS>

Dear Flow Group,

I have seen several publications lately that show BrdU cell cycle analysis, but show
both PI and FITC channels on a linear scale. I generally look at FITC on a log scale.
Does this affect results in any way?

Thanks for any comments.

Diane Sharp
DuPont Pharmaceuticals Company
Diane.M.Sharp at dupontpharma.com

From hsuematsu at hotmail.com Thu Apr 27 01:05:13 2000
From: hsuematsu at hotmail.com (Hotmail)
Date: Wed, 26 Apr 2000 23:05:13 -0700
Subject: anti IL-12 receptor beta
Message-ID: <20000427025741.71033.qmail@hotmail.com>

Hi, all
I'm looking for anti mouse IL-12 receptor beta 1 ,and 2.
Any information or suggestion may help.

Hidetoshi Takedatsu
research fellow
Immunopathology
Massachussetts general hospital

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From gap at MIT.EDU Wed Apr 26 17:10:00 2000
From: gap at MIT.EDU (Glenn Paradis)
Date: Wed, 26 Apr 2000 18:10:00 -0400
Subject: MIT flow cytometry position available
Message-ID: <v03020904b52cecbe8d12@[18.114.0.62]>




Hi Everyone,

We have an entry level Technical Assistant position available in the MIT
Flow Cytometry Core Facility located within the Center for Cancer Research.


The Facility is currently equipped with 2 FACScans, a FACS Calibur, a TSO
FACS Vantage, a FACStar Plus and a new yet to be determined high speed cell
sorter. The selected candidate will have a lot of responsibility including
daily operation and maintenance of a sorter, data analysis, data storage,
and scheduling. This person will work independently under the guidance of
the laboratory manager.

REQUIREMENTS: a B.S. in biological science and strong instrumentation
skills. Must be able to work well with diverse personalities. Experience
in flow cytometry and computer systems desirable, but will train the right
person. A two year commitment is preferred.
Job number 00-0282

To apply online go to http://web.mit.edu/personnel/www/resume.htm

MIT is an equal opportunity employer.

If you have any questions, please do not hesitate to email me.

Thank you,

Glenn Paradis
gap at mit.edu
617 253 6454










From joy.mundy at imvs.sa.gov.au Wed Apr 26 18:32:14 2000
From: joy.mundy at imvs.sa.gov.au (Joy Mundy)
Date: Thu, 27 Apr 2000 09:02:14 +0930
Subject: FW: 7AAD Trial
Message-ID: <000101bfafd7$a74660a0$8467140a@-romper.imvs.sa.gov.au>




-----Original Message-----
From: Janene White [mailto:janene.white at imvs.sa.gov.au]
Sent: Wednesday, 26 April 2000 16:53
To: cyto-inbox
Subject: 7AAD Trial


A researcher asks?

Can anybody help with a good explanation of staining with 7AAD then using a
cell fixative (paraformaldehyde) instead of PBS buffer. So, instead of using
the PBS (as in the
methodology) we are trialling the use of a cell fixative (post 7AAD
staining)
which will allow us increased flexability in work practice. It seems to be
quite successful (over a period of days the % of live v's dead remains
stable) except we are at a loss to explain the mechanics of how the
7AAD viability dye indicates that a percentage of cells are "live" when
theorectically they are all "dead" after cell fixation.

Janene Vine
Division of Human Immunology (Diagnostic)
Institute Of Medical and Veterinary Science
Frome Road, Adelaide 5000
E-mail : janene.white at imvs.sa.gov.au
Phone : (08) 82223476




From yves.st-pierre at inrs-iaf.uquebec.ca Wed Apr 26 16:19:23 2000
From: yves.st-pierre at inrs-iaf.uquebec.ca (Yves St-Pierre)
Date: Wed, 26 Apr 2000 16:19:23 -0500
Subject: Swine ICAM-1
Message-ID: <39075D3B.A3@iaf.uquebec.ca>


Dear Ed::

Cross-reactivity between ICAM-1 and its ligands has often been reported
in the literature. For instance, human CD11a/CD18 will bind murine
ICAM-1, or ICAM-2, for that matter, but not vice-versa. Binding of
swine neutrophils to human ICAM-1 on HUVECs is thus not surprising. But
this is very different from cross-reactivity at the level of the
antibodies. For instance, antibodies to human and mouse don't
cross-react.

Theoretically, there are several anti-human ICAM-1 antibodies that are
available commercially or that have been raised by private investigators
that could be used for a sandwich ELISA for measuring sICAM-1. But
since swine ICAM-1 has not been cloned yet, (at least to my knowledge),
it is difficult to predict if the human kit will work. The best thing
to do would be to try the human kit (it uses a polyclonal and a
monoclonal, if I remember well), but the incredibly unreasonnable price
of these kits makes you think twice before ordering it !!!

Good luck !

Yves

******************************
Yves St-Pierre
Professor
INRS-Institut Armand-Frappier
531 Boul. des Prairies
Laval, QC, CANADA H7V 1B7
Phone: 450-686-5354
Fax: 450-686-5501
E-mail: yves.st-pierre at inrs-iaf.uquebec.caEd Cabrera-EJ wrote:
>
> This is not directly related to cytometry, but perhaps someone in this
> conference has some insight into this issue.
> I am interested in measuring sICAM in swine plasma. There are kits available
> for human sICAM. Does anyone know if there is a good crossreactivity between
> human and swine sICAM. I have shown that neutrophils will adhere to HUVEC thru
> a mechanism that is inhibited by swine antiCD18, so I assume that there is a
> certain degree of crossreactivity since ICAM-1 is the main counterreceptor for
> CD18/CD11b.
>
> Any help will be appreciated.
>
> Thanks
>
> Ed Cabrera
> Procter and Gamble Pharmaceuticals
> Mason, OH


From bunny at cotleur.com Wed Apr 26 22:18:58 2000
From: bunny at cotleur.com (bunny)
Date: Wed, 26 Apr 2000 23:18:58 -0400
Subject: Cellquest/G4/OS9 update
Message-ID: <3907B19C.69E7CB6A@cotleur.com>


I know several posts have been made as to upgrades to make Cellquest
work on G4's/OS9. I just wanted to share my experience.
I just installed Cellquest 3.1 (not the latest version, by far) on my
new G4. Instead of installing off the original disks (no floppy drive
anyway) I used the backup CD I burned from my beige G3. I simply dragged
the Cellquest folder to the G4. Also copied ONLY eveinit (I still have
the ADB dongle)-none of the BD acquisition stuff. Plugged the dongle
into imate (have the 2.0x beta imate driver)- am in business!!

For those of you that only need analysis workstations- I've done this
method on every MacOS upgrade past 7.6. I always wondered if the crash
issues had to do with BDpac and the acquisition services. (I could be
wrong- but have not had ANY crashes from OS8.1/8.5/9.0, running yet!)

So maybe less is more- the older version Cellquest seems quite happy
with current MacOS!
And Adam- as soon as FlowJo does histogram overlays (the only thing I
use CQ for these days)- I'm there!




Bunny

BTW- CellQuest FLIES on a G4!




*******************************************************
Bunny Cotleur +*+ Bunny Cotleur
Cleveland Clinic Foundation *+* 2001 Lester RD
Neurosciences NC30 +*+ Valley City, OH 44280
9500 Euclid Avenue *+* 330-483-4800
Cleveland, OH 44195 +*+ bunny at cotleur.com
216-444-1164 *+*
cotleua at ccf.org +*+

*******************************************************
When you do something, you should burn yourself completely, like a good
bonfire, leaving no trace of yourself.
(Shunryu Suzuki)


From anardin at idm-biotech.com Thu Apr 27 01:29:13 2000
From: anardin at idm-biotech.com (Alessandra Nardin)
Date: Thu, 27 Apr 2000 08:29:13 +0200
Subject: Primary Conjugated antibodies against CD35 and CD55
Message-ID: <200004270629.IAA25045@william.bhdc.jussieu.fr>


Indeed, there are on average only 500 CR1 (CD35) molecules per human RBC,
which doesn't really make a bright direct staining easy.
I have been using home-conjugated mAbs for CD35 (therefore I don`t really
know what is available on the market) and I got a good pos./neg.
discrimination only with APC labelled mAbs (regardless of the specificity or
avidity of the mAbs).
Alternatively, you may try a biotinilated anti-CD35 + some kind of
conjugated avidin. This would allow you to do multicolor analysis and
should amplify the signal.

Alessandra





LUTI-IDM
Institut Biom?dical des Cordeliers
15, rue de l'Ecole de M?decine
75006 Paris

tel. +33 (0)1 53 10 17 75
fax +33 (0)1 53 10 17 80

http://www.idm-biotech.com

----------
>De?: "Jose A. Stoute" <stoutej at net2000ke.com>
>? : Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu>
>Objet?: Primary Conjugated antibodies against CD35 and CD55
>Date?: Mer 26 avr 2000 5:29
>

>
> Dear group, I am trying to setup assays of direct fluorescent staining
> of RBCs for CD35 and CD55. All the primary conjugated antibodies I have
> tried for these antigens that are commercially available give a very dim
> response, especially for CD35 which is known to have low expression on
> RBCs. I have had good results using indirect fluorescence using a
> secondary anti-mouse but this would preclude multicolor analysis, hence
> we would like to switch to direct fluorescence. Any suggestions as to
> which antibodies I could use or the approach?
>
> Thank you in advance for your assistance.
>
> Jose A. Stoute
>
> --
> Jose A. Stoute, MD
> Unit 64109, Box 401
> USAMRU-Kenya
> APO AE 09831-4109
> e-mail: stoutej at net2000ke.com
> Nairobi Tel 254-2-729303, Fax 254-2-714592
> Kisumu Tel 254-35-22942, Fax 254-35-22903
> Electronic Fax Service: 1-630-214-2008, 1-917-463-0373, 1-917-477-6048
>
>


From waddi002 at tc.umn.edu Wed Apr 26 15:16:07 2000
From: waddi002 at tc.umn.edu (Kevin Waddick)
Date: Wed, 26 Apr 2000 15:16:07 -0500
Subject: EMA -- continued
Message-ID: <39074E67.E806AC42@garnet.tc.umn.edu>


I posted the following message yesterday, however I forgot to mention
that I am using UV excitation of the EMA. That is because I am using up
all of the 488 nm PMTs on our FACS Vantage for 3 other fluorochromes.
The UV laser excites Hoechst 33342 and EMA.
I made up fresh EMA yesterday to compare it to the refrigerated EMA
made on 4/5/00 and tested them on freshly thawed patient cells that were
80-90% viable by trypan blue exclusion versus a cell line that had ~50%
viability. Surprisingly, considering what I saw before, the 5 ug/mL EMA
treatment yielded equivalent results using continuously growing versus
freshly thawed cells. When using 488 excitation, there was a clear
separation between live and dead (or EMA neg and pos, resp.) cells. The
separation when using UV excitation was not as good, but it was there if
one knew what they were looking for. I was also surprised to see that
the EMA that had been sitting in the refrigerator for approx. 3 weeks
gave either the same results as the freshly made EMA or only very
slightly worse. It appears that we did not have the Vantage set up
correctly before.
A question remains, however. Is there a way to get EMA to give stronger
emission when excited with the UV laser. As I said, the difference
between the EMA positive dead cells and the EMA negative live cells is
not as clear when the UV laser is used rather than the 488 laser. I
wouldn't have expected this, because when I used to assay apoptotic DNA
laddering on agarose gels the DNA was stained with ethidium bromide that
was excited with UV light. That fluorescence seemed quite bright to me.
I am sorry that my first message was misleading.

Kevin Waddick
------------------------------------------------------------------------
ORIGINAL MESSAGE

A week ago Howard Shapiro enlightened me (and maybe others) by
saying that, whereas propidium is (supposedly) membrane impermeant to
healthy cells, ethidium is membrane permeant and is pumped out of
healthy cells. Thus, propidium is a viability stain that operates by
passive dye exclusion and ethidium requires active participation on the
part of the healthy cell to appear negative for staining with this dye.
I am wondering if newly thawed cells have the ability to pump out
ethidium -- or, more specifically, ethidium monoazide (EMA) -- if they
are otherwise viable at that point. When I thaw cryopreserved cells I
get a clear-cut idea of their viability immediately afterward by trypan
blue exclusion (I realize that early apoptotic cells are trypan
negative) because it is a yes-or-no decision. When I stain with 5 ug/mL
EMA, leave the cells in the dark for 15 minutes and then expose them to
a nearby fluorescent light source for 15 minutes, I get a confusing
pattern when the cells are analyzed by flow cytometry after washing,
fixing, etc. Not even all of the cells obviously dead by FSC, SSC, and
hypodiploidy are positive for EMA staining. And the seemingly viable
cells have a wide range of EMA positivity, whereby many of them have
higher intensity than the lower intensity dead cells.
So, can EMA even be used right after cells have been thawed, or is
their permeability/dye pumping status totally messed up? I would like to
be able to quickly fix/permeabilize cryopreserved cells after thawing to
prevent further cell death and in preparation for intracellular staining
but be able to distinguish the cells that were dead prior to that point.

Also, is EMA unstable in solution at 4 C or RT, unlike plain old
ethidium bromide? The results I got soon after making a stock solution
of 0.5 mg/mL EMA were quite different from those 2 weeks later when the
stock had been sitting in the refrigerator.

Kevin G. Waddick, Ph.D.
Parker Hughes Institute
2657 Patton Road
St. Paul, MN 55113


From DAVISB at MAIL.MMC.ORG Thu Apr 27 13:21:03 2000
From: DAVISB at MAIL.MMC.ORG (Bruce Davis)
Date: Thu, 27 Apr 2000 14:21:03 -0400
Subject: Resume: monocyte marker
Message-ID: <s90857cd.080@mail.mmc.org>


I am surprised that everyone agreed on CD14, as CD64 would get my vote as the better
marker.



Bruce H. Davis, M.D.
Maine Medical Center Research Institute
125 John Roberts Rd., Suite #8
South Portland, Maine 04106

207-842-7914
FAX: 207-761-2130
Email: davisb at mail.mmc.org

>>> "robert gniadecki" <rgniadecki at hotmail.com> 04/27/00 09:00AM >>>

I would like to thank everybody who answered my question. Everybody agreed
that CD14 is the best macrophage/monocyte marker.
Thanks again,
Robert Gniadecki
Copenhagen
________________________________________________________________________
Get Your Private, Free E-mail from MSN Hotmail at http://www.hotmail.com




From COuma at kisian.mimcom.net Fri Apr 28 03:13:58 2000
From: COuma at kisian.mimcom.net (Ouma, Collins)
Date: Fri, 28 Apr 2000 11:13:58 +0300
Subject: Jurkat T cell clone
Message-ID: <A58AAEF23E44D311AFC600105A0B3AA902C107@cdckisian>


Hello
Has anyone used the Jurkat Clone E6-1 from ATCC? I wanted to know how it can
be diluted.
Regards
Collins.


From smonard at trudeauinstitute.org Thu Apr 27 13:54:28 2000
From: smonard at trudeauinstitute.org (Simon Monard)
Date: Thu, 27 Apr 2000 14:54:28 -0400
Subject: Macrophage Calcium flux
Message-ID: <s90854c0.027@trudeauinstitute.org>


Hi

We are having some problems doing calcium flux experiments (using Fluo-3 and Fura-Red)
with mouse peritoneal macrophages. Even with ionomycin not all the cells respond and
those that do, respond less than we would expect. Are such cells known to be hard to
load? Do they sequester the dyes into cytoplasmic compartments? We have done plenty
of successful experiments with other cell types.

Thanks for any insights



Simon Monard
FACS Lab Manager
Trudeau Institute
Saranac Lake
NY12983

Ph 518 891 3080 X352


Simon Monard
FACS Lab Manager
Trudeau Institute
Saranac Lake
NY12983

Ph 518 891 3080 X352


Simon Monard
FACS Lab Manager
Trudeau Institute
Saranac Lake
NY12983

Ph 518 891 3080 X352



From bunny at cotleur.com Thu Apr 27 23:46:45 2000
From: bunny at cotleur.com (bunny)
Date: Fri, 28 Apr 2000 00:46:45 -0400
Subject: FloJo clarification
Message-ID: <390917A5.E007DDE6@cotleur.com>


Fellow Flowers-

My comment about FloJo not capable of histogram overlays was a wee bit
off kilter.
Yes, FloJo DOES do fabulous overlays. (In fact, it's pretty cool that it
can overlay DOT PLOTS!)
What I failed to mention was my interestreally is in having K-S stats on
those overlays: I was looking for numbers, not the visual.
BUT! I've been told that K-S stats are just around the corner; so stay
tuned......



Bunny




*******************************************************
Bunny Cotleur +*+ Bunny Cotleur
Cleveland Clinic Foundation *+* 2001 Lester RD
Neurosciences NC30 +*+ Valley City, OH 44280
9500 Euclid Avenue *+* 330-483-4800
Cleveland, OH 44195 +*+ bunny at cotleur.com
216-444-1164 *+*
cotleua at ccf.org +*+

*******************************************************


From steve.woodard at ibb.gatech.edu Wed Apr 26 11:32:25 2000
From: steve.woodard at ibb.gatech.edu (Steve Woodard)
Date: Wed, 26 Apr 2000 11:32:25 -0500
Subject: Viability assay using flow cytometry
Message-ID: <4.1.20000426113046.00bd14a0@ibbmail.coon.gatech.edu>


Dear flowers,

What is the best way to perform a viability assay using flow cytometry? I
am currently using calcein AM and propidium iodide (PI). I substituted the
ethidium homodimer with PI only because PI appears to be a common indicator
of dead cells in flow cytometry and it is less expensive. For those of you
who have FACS Vantages, is it possible to compensate between FL1( Calcein)
and FL3 ( PI) in your systems?

For some reason, I do not see distinct populations of cells when I use the
previously mentioned stains. In fact, I see a population that appears to be
positive for PI and positive for calcein based on controls ( live cells:
calcein only and Etoh fixed cells: PI only).
All responses are appreciated.

Steve



From adam at treestar.com Thu Apr 27 23:53:34 2000
From: adam at treestar.com (Adam)
Date: Thu, 27 Apr 2000 21:53:34 -0700
Subject: FlowJo/G4/OS X update [was: Cellquest/G4/OS9 update]
References: <3907B19C.69E7CB6A@cotleur.com>
Message-ID: <3909194D.BAEADFAE@treestar.com>




bunny wrote:
>

> And Adam- as soon as FlowJo does histogram overlays (the only thing I
> use CQ for these days)- I'm there!
>
FlowJo does histogram overlays. See
<http://www.treestar.com/flowjo/v3/html/leoverlays.html>

I called Bunny, and she was referring to the Kolmogorov-Smirnoff
statisitic to perform histogram comparison. FlowJo will create and
export the Cumulative Distribution Functions (the integral of the
histogram) which is the basis of the K/S statistic, so you can easily
perform this test with the help of a spreadsheet. But we still don't
have that function built in. I have a prototype built, and you'll
likely see this feature included one day soon.

We'll probably get it into the OS X compatible release, due when Apple
releases the new operating system in the summer. But the tools we need
don't get released until next month, so its hard to predict the
schedule. Nevertheless, FlowJo worked with OS9 and the G4 the day they
shipped, and we'll endeavor to support OS X on that same timeframe.

>
> BTW- CellQuest FLIES on a G4!
>
Of course, that's relative to CellQuest on a G3. On the same equipment
with the same data, FlowJo outperforms CellQuest hands down.


Adam


-----------------------------------
Adam Treister
Tree Star, Inc.
www.flowjo.com
800-366-6045
-----------------------------------


From kukuru at umich.edu Fri Apr 28 07:59:16 2000
From: kukuru at umich.edu (Mark A. KuKuruga)
Date: Fri, 28 Apr 2000 08:59:16 -0400
Subject: BrdU/PI cell cycle analysis
References: <0221C39073FDB006M/@MHS>
Message-ID: <39098B24.EBC4D395@umich.edu>


Diane,
Some are of the opinion that log amplification of the BrdU labeling may be too sensitive,
thus revealing some low level, and possibly non-chromatin DNA, content. We've looked at
this both ways, and generally find the numbers to correlate. It is true that with a
direct labeling technique the signals are considerably lower than with an indirect
system,
so linear display of the BrdU is workable. If, however, your labeling technique gives a
very bright result, you may be forced to use a log display to manage it.
Again, it's probably a good idea to look both ways, and see for yourself if there are
significant differences.
MAK.

Diane M Sharp wrote:

> Dear Flow Group,
>
> I have seen several publications lately that show BrdU cell cycle analysis, but show
> both PI and FITC channels on a linear scale. I generally look at FITC on a log scale.
> Does this affect results in any way?
> Thanks for any comments.
>
> Diane Sharp
> DuPont Pharmaceuticals Company
> Diane.M.Sharp at dupontpharma.com

---
Mark A. KuKuruga, Managing Director
University of Michigan Core Flow Cytometry
<http://www.cancer.med.umich.edu/flow_cytometry>
phone: 734-647-3216 fax: 734-936-7376
kukuru at umich.edu




From ntflow at odin.mdacc.tmc.edu Fri Apr 28 15:14:24 2000
From: ntflow at odin.mdacc.tmc.edu (Nicholas Terry)
Date: Fri, 28 Apr 2000 15:14:24 -0500
Subject: BrdUrd/PI
Message-ID: <4.3.1.0.20000428151126.00af14d0@143.111.62.32>



>Dear Flow Group,
>
>
>I have seen several publications lately that show BrdU cell cycle
>analysis, but show
>both PI and FITC channels on a linear scale. I generally look at FITC on
>a log scale.
>Does this affect results in any way?
>
>
>Thanks for any comments.
If BrdUrd-labeled fluorescence is measured with logarithmic amplification
the effect is to make the marginal distribution approximately normal. This
(in my opinion) makes it much easier to discriminate labeled cells compared
with linear amplification. Others I know disagree.
Best wishes,
Nick
Nicholas Terry, M.A., Ph.D.,
Experimental Radiation Oncology - 066,
The University of Texas M. D. Anderson Cancer Center,
1515 Holcombe Blvd., Houston, Texas 77030.
'Phone: 713-792-3424,
'Fax: 713-794-5369.
http://drad52.mdacc.tmc.edu.
ntflow at odin.mdacc.tmc.edu.



From steve at habanero.cb.uga.edu Fri Apr 28 17:00:49 2000
From: steve at habanero.cb.uga.edu (Steve G. Hilliard)
Date: Fri, 28 Apr 2000 18:00:49 -0400 (EDT)
Subject: Mycoplasma anyone?
Message-ID: <Pine.LNX.4.10.10004281746440.7356-100000@habanero.cb.uga.edu>


Hi folks,

I was just approached by a student who is looking at Mycoplasma
pneumoniae, and he's got an interesting question. He has a mutant that
seems to have an altered chromatin condensation, which he has been looking
at using image analysis, but he would like to use flow. His
approach is to stain the DNA w/ DAPI, and the membrane with another
dye, and then analyze the area associated with each color. The ratio
of nucleoid area to total cellular area differs btwn wild type and
mutant.

He considers the microscopy a pain, and would like to use flow, but
this type of spatial analysis is the type of thing that imaging is perfect
for. For one thing, these things are tiny, and bad about clumping.
Has anyone had any success with M. pneumoniae or relatives in a flow
system?? Should I be giving this guy a pitch for a laser scanning
system?

Any suggestions welcomed,
Steve
+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
Steve G. Hilliard (706) 542-9474
University of Georgia Cell Analysis Facility
flowman at uga.edu http://floweb.cb.uga.edu/



From jwright at uoguelph.ca Fri Apr 28 13:43:23 2000
From: jwright at uoguelph.ca (Jennifer Wright)
Date: Fri, 28 Apr 2000 14:43:23 -0400
Subject: cryopreservation of lymphocytes, later to be used in flow cytometry
Message-ID: <3909DBCB.A9E88B2C@uoguelph.ca>


Does anybody know of (or uses) a method for the isolation,
cryopreservation, and thawing of lymphocytes (in particular equine
lymphocytes)? These cryopreserved lymphocytes are to be later analyzed
using the flow cytometer.

Thanks
Jen


From esimons at bu.edu Fri Apr 28 14:44:57 2000
From: esimons at bu.edu (Elizabeth R. Simons)
Date: Fri, 28 Apr 2000 14:44:57 -0500
Subject: Resume: monocyte marker
References: <s90857cd.080@mail.mmc.org>
Message-ID: <3909EA39.1EFB8BFF@bu.edu>


Actually, as we've published (Bernardo et al, J. Leuk.Biol.), naive circulating
monocytes as well as those incubated for 24-96 hrs and hence monocyte-derived
macrophages do not all express CD 14. Those that are CD14 positive are more responsive
(e.g. with a Ca++ spike) than those which are negative for CD 14, even when all are
clearly monocytes. Therefore CD14 is not a good marker for monocytes in general.
Elizabeth R. Simons, Ph.D.
Professor of Biochemistry
Boston University School of Medicine
80 E.Concord St.
Boston, MA 02118


Bruce Davis wrote:

> I am surprised that everyone agreed on CD14, as CD64 would get my vote as the better
> marker.
>
> Bruce H. Davis, M.D.
> Maine Medical Center Research Institute
> 125 John Roberts Rd., Suite #8
> South Portland, Maine 04106
>
> 207-842-7914
> FAX: 207-761-2130
> Email: davisb at mail.mmc.org
>
> >>> "robert gniadecki" <rgniadecki at hotmail.com> 04/27/00 09:00AM >>>
>
> I would like to thank everybody who answered my question. Everybody agreed
> that CD14 is the best macrophage/monocyte marker.
> Thanks again,
> Robert Gniadecki
> Copenhagen
> ________________________________________________________________________
> Get Your Private, Free E-mail from MSN Hotmail at http://www.hotmail.com



From royal011 at visto.com Sat Apr 29 19:33:13 2000
From: royal011 at visto.com (Bratislav Janjic)
Date: Sat, 29 Apr 2000 17:33:13 -0700
Subject: non-specific binding and mitochondria staining
Message-ID: <200004300033.TAA21695@flowcyt.cyto.purdue.edu>


Hi,

How can I block non-specific binding in procedure of purification B cells from PBMNC
using negative immunoselection by immunomagnetic beads. For that purpose, I tried to use
different concentrations of human Ig (to block Fc receptors), but still, I have got only
5-6 million of B cells from 200 million PBMN cells because of non-specific binding. By
the way for this purpose I use anti-CD3, CD5, CD56, CD16, CD14 CD13 and anti-glycophorin.

My next question is about ApoAlert mitochondrial detection kit by flow-cytometry
(Clonitech company), and MitoSensor, dye that company gives in this kit for mitochondrial
staining, that is used for determination of ��healthy�� mitochondria and mitochondria that
are changed in the process of apoptosis. I would like to know what is the experience
of people that use this kit, because the company provide only technical support for
this kit, but not the examples of results that were got.

Sincerely,
Bratislav Janjic, B.Sc.
Department of Pathology E1005
Biomedical Science Tower
University of Pittsburgh Cancer Institute
PA 15213



__________________________________________________________________________
Visit http://www.visto.com/info, your free web-based communications center.
Visto.com. Life on the Dot.



From royal011 at visto.com Sat Apr 29 10:18:02 2000
From: royal011 at visto.com (Bratislav Janjic)
Date: Sat, 29 Apr 2000 08:18:02 -0700
Subject: Non-specific binding and mitochondria staining
Message-ID: <200004291518.KAA20019@flowcyt.cyto.purdue.edu>


Hi,

How can I block non-specific binding in procedure of purification B cells from PBMNC
using negative immunoselection by immunomagnetic beads. For that purpose, I tried to use
different concentrations of human Ig (to block Fc receptors), but still, I have got only
5-6 million of B cells from 200 million PBMN cells because of non-specific binding. By
the way for this purpose I use anti-CD3, CD5, CD56, CD16, CD14 CD13 and anti-glycophorin.

My next question is about ApoAlert mitochondrial detection kit by flow-cytometry
(Clonitech company), and MitoSensor, dye that company gives in this kit for mitochondrial
staining, that is used for determination of ��healthy�� mitochondria and mitochondria that
are changed in the process of apoptosis. I would like to know what is the experience
of people that use this kit, because the company provide only technical support for
this kit, but not the examples of results that were got.

Sincerely,
Bratislav Janjic, B.Sc.
Department of Pathology E1005
Biomedical Science Tower
University of Pittsburgh Cancer Institute
PA 15213


__________________________________________________________________________
Visit http://www.visto.com/info, your free web-based communications center.
Visto.com. Life on the Dot.



From gap at MIT.EDU Fri Apr 28 17:21:51 2000
From: gap at MIT.EDU (Glenn Paradis)
Date: Fri, 28 Apr 2000 18:21:51 -0400
Subject: Viability assay using flow cytometry
In-Reply-To: <4.1.20000426113046.00bd14a0@ibbmail.coon.gatech.edu>
Message-ID: <v03020901b52fbe32d0d0@[18.114.0.62]>


Hi Steve,

Here is a quick response to compensation issue between Fl1 and Fl3 on a
Vantage. Just take the signal lead (not the black voltage lead) off the
Fl3 PMT and put the signal lead from the Fl2 PMT on your Fl3 PMT. Your FL3
data will be routed to your Fl2 electronics and thus you can now do Fl2-%
Fl1 compensation. To display your data put up a dot plot of Fl1 vs Fl2.

Take care.

Glenn
MIT


>Dear flowers,
>
>What is the best way to perform a viability assay using flow cytometry? I
>am currently using calcein AM and propidium iodide (PI). I substituted the
>ethidium homodimer with PI only because PI appears to be a common indicator
>of dead cells in flow cytometry and it is less expensive. For those of you
>who have FACS Vantages, is it possible to compensate between FL1( Calcein)
>and FL3 ( PI) in your systems?
>
>For some reason, I do not see distinct populations of cells when I use the
>previously mentioned stains. In fact, I see a population that appears to be
>positive for PI and positive for calcein based on controls ( live cells:
>calcein only and Etoh fixed cells: PI only).
>All responses are appreciated.
>
>Steve





From snezna at nmt.edu Fri Apr 28 17:38:30 2000
From: snezna at nmt.edu (Snezna Rogelj)
Date: Fri, 28 Apr 2000 16:38:30 -0600
Subject: Practical Flow Cytometry
Message-ID: <007301bfb162$7aa7ec80$320e8a81@nmt.edu>

Greetings,
Does anyone have an extra/old copy of Shapiro's FlowBible (Practical Flow Cytometry) that they are willing to sell? The publisher Wiley says they are out of stock and that the delivery date is uncertain.
We are in a desperate need for the wisdom within those pages....
Thank you, Snezna

Snezna Rogelj, Ph.D.
Biology Department
Jones Annex 315
New Mexico Institute of Mining and Technology
Socorro, New Mexico 87801
Phone & FAX: 505-835-5608
Email: snezna at nmt.edu
-------------- next part --------------
HTML attachment scrubbed and removed

From Roederer at drmr.com Fri Apr 28 19:02:21 2000
From: Roederer at drmr.com (Mario Roederer)
Date: Fri, 28 Apr 2000 17:02:21 -0700
Subject: Job Opportunity: VRC flow cytometry facility manager
Message-ID: <v04220802b52e40e63816@[128.218.108.229]>


(For more information, please see <http://www.drmr.com/vrc/>).

We are seeking an experienced and independent individual to manage
the new Vaccine Research Center (VRC, NIAID, NIH) flow cytometry
facility. The core facility will contain an analyzer as well as two
high-speed sorters. One sorter will be operational in the building's
P3 biohazard facility; the other will be capable of up to 12-color
analysis. The core facility will be located on the top floor of the
new Building 40 on the NIH Bethesda campus
(<http://www.niaid.nih.gov/vrc/vrcslide/sld006.htm>). Duties will
include overseeing installation, optimization and routine operation
of the instruments, as well as providing training and technical
support for core operators and users. In addition, this individual
will assist in the development and optimization of high-throughput
FACS based immune assays targetted for preclinical or clinical
vaccine trials, including the application of 12-color
immunophenotyping/functional assays. The successful applicant should
have at least 10 years experience with flow cytometry including
coordinating a multi-user facility, and preferably an M.S., Ph.D., or
M.D. (although exceptionally-qualified candidates with other degrees
will be considered). In addition, the applicant should have
experience in designing, executing, and analyzing immunophenotyping
and immune functional assays with little or no supervision.
Excellent interpersonal skills are essential. Salary will start at
$61,000 - $85,000/year, depending on experience and qualifications.
Applicants should send a curriculum vitae, bibliography, statement of
research experience and career directions, and three letters of
reference to:

Mario Roederer
521 Parnassus Ave, Room C634C
San Francisco, CA 94143
415-514-0395
FAX 415-476-4204
EMail: Roederer at drmr.com

Submission of materials by EMail is encouraged. Applications must be
received by June 1, 2000.

The NIH is an equal opportunity employer.


From Roederer at drmr.com Fri Apr 28 19:02:10 2000
From: Roederer at drmr.com (Mario Roederer)
Date: Fri, 28 Apr 2000 17:02:10 -0700
Subject: A change of venue
Message-ID: <v04220807b52fce04831b@[63.195.112.129]>


Dear Flow Fans, Flow Enthusiasts, and FlowJocks:

I have recently been successfully recruited by Gary Nabel and the new
Vaccine Research Center at the NIH--to move my laboratory as well as
to create a new advanced flow cytometry core facility serving the VRC
and the NIH. This means that I'll be hiring several new people, both
for the core facility as well as for my lab, in the coming
months--I'll send announcements as the positions become available.

I'd like to encourage anyone who is interested to look at the web
pages (<http://www.drmr.com/vrc/>) to get a feel for what my lab and
the core facility for the VRC will be doing. I'm going to try to set
up the facility to be different than most core facilities that are
simply service facilities. Instead, I'd like to complement the other
facilities at NIH by providing a unique, state-of-the-art, advanced
facility. We'll have high-speed P3 sorting, a 12-color capable
machine, as well as standard instrumentation. We'll also have
fluorescence microscopy, laser capture microdissecting microscopes,
and will acquire other necessary technology in the future.

I'm looking specifically for staff that will work with users to train
them them to use machines, who can interact with biologists, and can
become an integral part of the experimental process! This means I'm
particularly interested, for the core facility, in experienced flow
cytometrists who also have a strong desire to participate in doing
the experiments, in developing new assays and new technologies, and
in making sure that users understand what it is they're trying to do!
(OK, with regard to the last point: I'm an idealist...)

In addition, I'll have several additional slots for postdocs in my
new laboratory. I encourage any graduate students or current
postdocs (I have the flexibility to hire senior scientists, if
warranted) to look over the research plans and to propose projects
that complement current interests, or to work on the current
projects. Considering the enormous interest from NIH scientists in
collaborating to use the 12-color technology, there's no shortage of
possible projects.

The resources at the NIH are unequalled anywhere in the world, and
the VRC, which is moving into an entirely new building on the campus,
will reap the benefit of these enormous resources. I am very excited
about this move--which, incidentally, happens in August.

Please pass on this note to others that you think would be interested
in these opportunities. I appreciate any advice and help from the
community as I try to put together an outstanding and productive
research lab and facility!

Sincerely,

Mario Roederer, Ph.D.
Director, Flow cytometry core facility
Vaccine Research Center, NIH

VRC info and jobs: <http://www.drmr.com/vrc/>
Home page: <http://www.drmr.com/>


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