Jan I don't see annexin binding to HL-60 treated with camptothecin (CAM), even though other assays show a significant (>50%) apoptotic population (see CYTOMETRY 28:253). I have heard from other people who see similar things with HL-60. However, I have also heard from people who say annexin works with HL-60 + etoposide (nobody has yet told me that annexin works with HL-60 + CAM). I am not clear as to whether there are different HL-60 sublines which do or do not bind annexin during apoptosis or whether different stimuli produce different results with respect to phosphatidylserine. Tom _______________________________________________________________________________ Subject: Annexin V and Ca++? From: Jan Vondracek <hivrisek@ibp.cz> at INTERNET Date: 1/26/98 3:43 PM I am a complete novice in this field. I work mostly on HL-60 cells. I just started to use annexin-V-FITC to detect apoptotic cells and I am not very happy with results. In fact, I can detect only about 1% of apoptotic cells in my positive control (treated with etoposide), while I know that there should be around 30% (determined morphologically under microscope and judging from sub-G1 peak).I get only small double positive and double negative (over 90%) populations. I know that various producers of annexin recommend different buffers with different CaCl2 concentrations. Could this be the reason of poor annexin staining? My second question is perhaps trivial. Why not to use FL3 to detect PI positive cells when discriminating apoptotic and necrotic cells - I could avoid any compensation error? Is there any problem that I do not know about? Thanks for your comments. Jan Vondracek hivrisek@ibp.cz Institute of Biophysics Kralovopolska 135 612 65 Brno Czech Republic
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