>I also have a question: I see a more heterogeneous side scatter profile >in the activated cells [lymphocytes]. How generous should a CD3/ side scatter gate be >to include all relevant events? > That depends in part on whether you believe your CD3 staining. In my experience, separation between CD3-positive and negative cells deteriorates somewhat with activation, and it isn't always clear whether some of the cells with higher side scatter are positive or negative. Based on multiparameter analysis using DNA and RNA stains as well as fluorescent antibodies for phenotype and activation status, there seem to be substantial numbers of both apoptotic cells and aggregates (doublets, triplets, etc.) in stimulated cultures, and at least some of these contribute to the high side scatter population. Again, whether you consider them relevant is a matter of opinion. I've been trying to find an answer to your question for years, and I'm not sure I'm any closer than I was when I started. -Howard
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