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=