Hi Slava, I have worked with flow cytometry to detect cytokine production
after 6 hours stimulation of whole blood with PMA and ionomycin/brefeldin
A. The system work very well and we always get a large amount of IFN-gamma
producing T cells. However when we work with antigen-specific stimulation,
the amount is very small in comparison with PMA, but still higher than
unstimulated controls. We have followed the conventional protocols listed
in the literature for PMA stimulation. With the antigen, we have used
1 hour of previous stimulation and then add the brefeldin A. All the
reagents we use are from SIGMA (PMA, Ionomycin and BrefeldinA) and
the monoclonals for flow cytometry are from BD (surface marker-FITC
labelled) and pharmigen (Cytokines-PE labelled). For permeabilization
we have used fixation with Lysing solution (BD or coulter) followed by
saponin treatment.
Good Luck
Slava wrote:
----------
From: Slava Epelman[SMTP:sepelman@ucalgary.ca]
Sent: Sexta-feira, 8 de Janeiro de 1999 18:49
To: cyto-inbox
Subject: IFN-g detection
Hi,
I was wondering if anyone has looked at IFN-g production by
flow cytometry? I am able to detect TNF-a from monocytes, but I
can't detect any IFN-g from T cells or NK cells (even though I have
detected
protein secretion by ELISA). Any thoughts?
Slava Epelman
University of Calgary
sepelman@acs.ucalgary.ca
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