I would like to TCR type a population of human antigen specific CD4 T cells detected using intracellular cytokine staining. I am staining after fixation, under which conditions I find 90-95% of mAbs "work". Questions: 1. Using commercially available anti-Vbeta mAbs, what % of the TCR repertoire is "covered". 2. Is this type of analysis realistic or an experimental black hole? 3. Any suggestions for anti-Vbeta mAbs, particularly panels that have been worked out? 4. Any suggestions for "tricks" to make this easier? One maneuver I am considering is to pool a group of 4 anti-TCR mAbs for each initial screening. That way I could cover the available repertoire in 4-5 tubes. In those that are positive I would then go back and look at the 4 Vbetas individually. 5. References? Thanks, Calman
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