Re: counter-stain surface CD3 and intracellular CTLA-4

Hi!

Two things:

I have seen this phenomenon of change in FSC SSC as well after
permeabilizing cells. That might be an artefact of the treatment, after all
you punch wholes in the cell membrane!

A way to get around the problem of having a "wrong gate" with your subset
stain is to permeabilize the cells in all the other tubes as well (after the
initial stain). Also, do you have a surface stain control for your CTLA-4?
To be able to say how much of the staining you get on your permeabilized
sample is actually due to surface instead of intracellular staining?

Cheers, Anja

> --- Ursprüngliche Nachricht ---
> Von: erfanin@sums.ac.ir
> An: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
> Betreff: counter-stain surface CD3 and intracellular CTLA-4
> Datum: Sun, 11 Sep 2005 10:11:43 +0430 (IRDT)
> 
> Dear flowers
> 
> We are trying to counter-stain surface CD3 and intracellular CD152 (CTLA4)
> on/in lymphocyte isolated from lymph node. After isolating the cells on
> density gradient and dissolving them in PBS 1X with 0.1%    sodium azide (
> Staining/Washing (S/W) solution) , we perform surface two-color staining
> for several molecules in several tubes including one for leukogate
> (CD45/CD14).Another tube, designed for the above mentioned purpose, is
> stained for CD3 in this stage. Incubating for 20 minutes in dark and then
> washing with S/W solution, the first tubes are then fixed with BD fixative
> and store in refrigerator before analyzing in 24 hours. These tubes have
> acceptable results and normal lymphocyte ranges and plots.
> In order to permeabilize the cells in  the last tube (surface-CD3
> stained)we add 0.5 ml Saponin buffer (0.5 % Saponin in 1X PBS and 0.1 %
> Sodium azide) or BD FACS Permeabilizing Solution-2 and incubate for 10
> minuets at room temperature . Washing and dissolving the cells in 0.1 ml
> of S/W solution, Ab to CTLA-4 are then added and incubate for 20 minutes
> in dark. The cells are then washed with S/W solution and fixed in BD
> fixative and refrigerate before analyzing in 24 hours.
> In contrary to other tubes the cells in this tube seems to go smaller with
> low FSC and accumulate around and/or lower the Chanel 50 (256-Chanel
> scale). The cells are so small that fall out of the gate set according to
> leukogate tube (surface CD45 and CD14).
> I'm a beginner in FCM.	Any suggestion and comment might help me and would
> be appreciated.I would be glad if some one address me a standard protocol
> for counter-staining surface CD3 and intracellular CD152.
> 
> Thanks in advance.
> --
> N Erfani
> PhD student of Immunology
> Department of Immunology
> Medical School
> Shiraz University of Medical Sciences
> P.O. Box: 71345-3119
> Shiraz-Iran
> E-mail: erfanin@sums.ac.ir
> Tel: +98(0)711 2303687
> Fax: +98(0)711 2304952
> 
> 
> 
> 
> 

-- 
_______________________________________________
Anja Scholzen
PhD Student

The Austin Research Institute
Vaccine Development and Infectious Diseases Unit
Studley Road, Heidelberg 3084
VIC, Australia
Tel.:0061 3 9287 0673
Fax: 0061 3 9287 0600

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