Hi Jan, HL60 is a nice cell line for checking whether your apoptotis detection system is working, and I have used these cells successfully with the Annexin technique. As you say you are seeing a sub-G1 peak, I would also expect you to see a significant amount of apoptotic and dead cells by the Annexin technique. To induce apoptosis I would usually use 50ug/ml etoposide or 100ug/ml camptothecin for about 4 hours. Also dont forget that you are looking at a "snapshot" of the cells at the particular moment of analysis and the percentages of 'apoptotic' cells found by different methods of analysis may not be identical. The binding of Annexin to externalised PS residues is calcium-dependent, but if you are using a kit, the buffers supplied should be OK. If not I would use a buffer of 10mM HEPES, 140mM NaCl and 2.5mM CaCl2 (pH7.4). Wash your cells in this prior to the Annexin staining (Annexin at a concentration of 2.5ug/ml). As for your other question, you could detect PI in the FL3 channel, but there will still be a slight compensation problem of the PI into FL1 and also the FITC into FL3. This can be compensated but it may not be immediately obvious how to do it (depending on your machine). And certainly if you wanted to use FL2 for a PE label you would encounter difficulty. Hope this helps! Derek On Mon, 26 Jan 1998, Jan Vondracek wrote: > > 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? **************************************************************************** * Derek Davies Voice: (44) 0171 269 3394 * * FACS Laboratory, FAX: (44) 0171 269 3100 * * Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk * * London, UK * * * * Web Page: http://www.icnet.uk/axp/facs/davies/index.html * ****************************************************************************
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