I am facing a problem with the procedure for estimation of intracellular cytokines from whole blood.I am following the procedure in the Pharmingen catalogue. I dilute blood in RPMI medium and addPMA and A23187 at 50ng/ml and 1ug/ml to stimulate the cells and Brefeldin A at 1ul/ml (Golgiplug)The problem is that the highest signal I am getting is in uninduced controll cells but stimulatedblood does not give me a major fold difference in fact it is less than unstimulated blood. Can some one pl tell me what is happening?This I have done twice and both the times the result is the sameThe only thing that is comming to my mind is that some how stimulated cells are getting depleted during the treatment procedure and so giving less signal. Is it possible that the stimulated cells are becoming more fragile ? Also the CV is consistenty high > 300. The machine is a FACs Vantage and set using FITC Beads.
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