Re: BD fastimmune protocol
Howard Shapiro (hms@shapirolab.com)
Wed, 26 Feb 1997 18:47:49 -0500 (EST)
>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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