Following up on my previous general rare event question - thanx to all who responded. Now I have the specifics of the experiment and need your help. The investigator is looking for gamma delta TCR cells which are CD45 positive, usually CD4 neg, frequently, but not always, CD8 positive. 95% of Tcells are alph beta TCR positive and only 0.5-5% are gamma delta positive. He is looking for these cells in mouse spleen and peripheral blood and is looking at using Pharmingen mabs. First we need to find the cells and ultimately he wants to phenotype them as to CD4/CD8a or CD8b. This is what we are considering. CD45 FITC for lymphcyte gate (could also use FCS and SSC, but this is better?) B220 PerCP to exclude B cells beta TCR APC to exclude alpha beta cells gamma delta PE to identify rare events (PE cause it's brighter than FITC) Later we can substitute CD4, CD8a or CD8b FITC (the 8b is only available in FITC and PE so this determined the FITC choices) instead of CD45 for phenotyping, assuming the scatter gate does as well as the CD45 gate. We are not using CD3 to identify Tcells because it is my understanding that in mice CD3 is not really good at identifying all the Tcells, correct? I need help with a) is my logic sound, or would you suggest other marker combinations? and b) what is the gating process you would use to do this? Again, any and all suggestions are greatly appreciated.
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