Dear flowers- I am somewhat perplexed. I routinely perform intracellular cytokine staining on rhesus PBMC where we stimulate with specific antigen (SIV peptides in our case) to look for Ag-specific T cells responses (both CD4 and CD8). The protocol works extremely well. A colleague asked me to perform intracellular staining on mouse splenocytes using the two peptides which she immunized her mice with. Try as I might I cannot get antigen-specific IFN-gamma production using my usual protocol (although with mouse-specific Abs instead of rhesus-specific). I searched the mailing list archives but could only find examples where people stimulate with PMA/Ionomycin not with specific antigen. Can anyone tell me: 1) if anyone else has tried antigen-specific intracellular staining for cytokines in the mouse; and 2) why people just use PMA and not specific antigen in their mouse assays These may be really dumb questions- I'm just not used to working with anything except primates! Any advice would be greatly appreciated, Sincerely, Helen Helen Horton PhD Wisconsin Regional Primate Research Center 1220 Capitol Court Madison WI 53715 Tel: (608) 265 3381 Fax: (608) 263 4031 hhorton@primate.wisc.edu
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