From: Brian Rabinovich (brian.rabinovich@utoronto.ca)
Date: Tue May 28 1996 - 02:37:53 EST
Addressed to all those who may be able to aid in the process of staining for intracellular cytokines using fluorescently tagged mAbs for FACS analysis on peripheral blood: I am currently performing an assay involving intracellular staining of cytokines from peripheral human blood to assess TH1 and TH2 populations of CD4+ cells (The protocol I am using is below) . I am currently NOT stimulating the blood after obtaing it as we wish to assess any possible alterations in TH1:TH2 between controls and patients with vascular disease in as pure a fashion as possible. I am finding, however, that unstimulated CD4+ cells from peripheral blood stain very poorly for both IL-4 and IFN-gamma, in most cases. I was hoping that someone might have a simple protocol for stimulating CD4+ cells to synthesize IL-4 or IFN-gamma depending on their phenotype so that I can stain for a proportionally amplified cytokine profile. Also, because we are drawing blood from patients and controls on a regular basis, I am looking for a stimulation procedure which is as simple and least time consuming as possible. Please help.....Brian A. Rabinovich, Research Institute, The Wellesley Hospital, University of Toronto. E MAIL: brian.rabinovich@utoronto.ca P.S. Please forward this mesage to anyone else you think could lend a hand
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