From: Raffi Manoukian (MANOUKIAN@A1.TCH.HARVARD.EDU)
Date: Thu Jul 02 1998 - 15:11:01 EST
Hi flowers, Has anybody used pharmingen's cd95(clone DX2) to mediate apoptosis in human peripheral blood lymphocytes? My problem is that my control cells (untreated and IgG1 treated) have the same level of apoptosis as the treated cells. I've tried 6hr incubations as well as 24hr incubations with the anti-fas, and I can never seem to get more apoptotic cells than the background(which is sometimes as high as 40%) . I've also tried stimulating the cells in culture before adding the anti-fas, with ConA or OKT3 for various time points(24, 48, 72hrs, even 6 days) in hopes of activating the cells to express more Fas, but still no difference. Should I be using a different clone like ch-11 or should I be priming the cells a different way if at all? By the way I am using annexin v-fitc/PI to detect apoptotic and dead cells. Any suggetions would be greatly appreciated. Raffi
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