Re: intracellular cytokine staining for IFNg

Hi Brett,

do you have a period at the beginning of the stimulation without 
Brefeldin? Just try PMA/iono add BrefA after two hours and incubate 
for another 4 hours. That should do the job. 
Still problems? Maybe you find an answer on 
http://icstaining.schuitemaker.info ?

Regards,

Joost
__________________________________________

J.H.N.Schuitemaker
Laboratory manager / Senior research technician
Cellular Immunology Group
Dept. Cell Biology & Histology
Academic Medical Center
P.O.box 22700
1100 DE Amsterdam
The Netherlands

Tel. + 31 20 5664960
Fax. + 31 20 6974156

http://www.amc.nl
http://icstaining.schuitemaker.info

----- Original Message -----
From: "Leav, Brett A" <BLeav@tufts-nemc.org>
Date: Thursday, May 12, 2005 5:57 pm
Subject: intracellular cytokine staining for IFNg

> 5/12
> hello 
> has anyone had any recent success with intracellular cytokine 
> staining for IFNg on murine
> lymphocytes?	i am trying to detect IFNg on murine splenocytes and 
> intestinallymphocytes.  using 5 hours PMA/ionomycin/brefeldin i 
> get good signal for TNFa (30% total
> splenocytes) and less good IL-2 (10%), but no IFNg signal.  my 
> antibody (XMG 1.2) stains
> the positve control cells (MiCK) from pharmingen nicely so i know 
> it is not a staining
> problem.  i even detect IFNg in 48 hour supernatants from 
> stimulated cells.  i just can't
> figure out why i can't detect the cytokine intracellularly.
> 
> thanks 
> 
> Brett A. Leav, MD
> Division of Geographic Medicine and Infectious Diseases
> Tufts-New England Medical Center, Box 041
> 750 Washington Street
> Boston, Massachusetts 02111
> office: (617) 636-2869
> fax: (617) 636-5292
> email: bleav@tufts-nemc.org
> 
> 
> 
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