I have used 50 ng/ml PMA and 250 ng/ml ionomycin. This works well for
stimulating baboon PBMC to synthesize IFN-gamma (overnight
stimulation with brefeldin A). I used Pharmingen's 4.SB3 interferon
gamma antibody, which for me works equivalently on rhesus and baboon
PBMC. For rhesus I have been using 2 micrograms/ml PHA (Murex, HA16)
+ CD28 mAb ( 1 microgram/ml Pharmingen, clone CD28.2), which will
yield a lower frequency of positives, compared to ionomycin + PMA, but
the positive cells are probably just as brightly stained for
IFN-gamma. I have no experience with IL4 intracellular staining.
Gib Otten
Vaccines Research, Chiron Corp.
______________________________ Reply Separator _________________________________
Subject: Cytokine Activation
Author: "Echeagaray; Patricia L." <echeagaray@serquest.com> at SMTP
Date: 7/12/99 9:53 AM
Greetings!
I am trying to stain for intracellular IFN-gamma and IL-4 in rhesus. Does
anyone have any suggestions as to concentration of PMA and ionomycin which
work as activators?
This activation step is not done in flasks, but is activation of 100 ul
whole blood (plus 100 ul of tissue culture media therfore a total volume of
200 ul). I tried to activate 24 hours and got absolutely nothing! (These are
however, normal cells, and I only used IFN -gamma this time as the IL-4 is
normally lower....or so I was told)
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