Hi Marc If you get no A-V staining, since anti-CD95 should work, there may be the problem of no Calcium in the media. ( I hope I don't insult your intelligence, but this has happened to me and a couple of other people). Once the Ca++ is in, the A-V is on. I believe there was a discussion about this a while ago in this list and I remember some good suggestions on there. Alternatively, you can also coat wells with CD95, rather than using Protein G. (Cover wells with 10ug/ml CD95 in PBS for about two hours and then take off PBS-CD95 (you can re-use this solution a couple times), then wash with complete media. After the wells are coated you can incubate your cells for the same time as you do with the Protein-G--CD95 aggregates. Hope this helps Artur > ---------- > From: Marc Jacobsen > Sent: Monday, November 15, 1999 13:09 > To: Cytometry Mailing List > Subject: Induction of apoptosis > > > Dear flowers, > > I`d like to induce apoptosis in PBMCs using monoclonal antibodies against > CD95 (clone DX2) and Protein G. I used freshly thawed PBMCs and IL2 > activated LAK-cells but until now failed to get cells positively stained > with Annexin-5-FITC. Even though I tried different antibody concentrations > (5-20µg/ml) and different incubation periods (4-24h) no apoptosis was > induced. > I would be very grateful if anyone could help me. > > Marc > > > > Marc Jacobsen > Klinische Neuroimmunologie > MZ Nervenheilkunde - Neurologische Klinik mit Poliklinik > Rudolf-Bultmann-Str.8, 35039 Marburg, Germany, > Fax +49 6421 28 65480, Phone +49 6421 28 65480 > >
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