Dear Dr Zucker I think that the discuss should be: What is the best approach to detect apoptosis in my investigation?? I tested annexin, tunnel and cell cycle. In my investigation, I found that the best way is by cell cycle (using PI). In my case, where macrophages is abundant, early apoptotic cells (T cells- in my investigation), expressing phosphatidylserine (PS), are rapidly phagocited (so annexin is not possible). Other problem in apoptosis, by annexin, is the time in apoptotic pathway. It's necessary to detect in very early stage, when apoptotic cells express PS but the cell membrane is impermeable to PI (so apoptotic cells are PS positive/PI negative cells). Tunnel is very interesting to use in histochemistry assay cause there is no significant differences between tunnel and cell cycle assay in suspension cells (see Journal of Immunological Methods 188:219-228). Conclusion: Analyse your apoptosis: Is strong that could be detect fast?? or it's weak that could be detect after some hours?? If fast, I suggest to try annexin. If yours cells delay to entry in apoptosis, try to detect by a DNA fragmentation method (like tunnel or cell cycle). Certify if have macrophages or not (when I deplete macrophages, I get better results) I hope that this observation could be useful... Best regards Victor Brasil -------Original Message------- From: Zucker.Robert@EPAMAIL.EPA.GOV Date: terça-feira, 02 de abril de 2002 17:01:39 To: cyto-inbox Subject: Apoptosis detection A colleague asked a simple question regarding a continous lymphoblast system exposed to toxicants. What is the best assay or kit to detect apoptosis? After a short discussion on flow assays that included --permeability endpoints, annexin, tunnel, and subg1, he decided on using the gold standard of laddering. He wants a definitive test. I thought there were more sensitive and more accurate flow cytometic assays to detect apoptosis. I also told him he usually needs two tests to make a conclusion reegarding apoptosis. Any opinions on what is the best and easiest approach to detect apoptosis in a suspension cell culture? Bob Robert M. Zucker, PhD U.S. Environmental Protection Agency Office of Research and Development National Health and Environmental Effects Research Laboratory Reproductive Toxicology Division, MD 72 Research Triangle Park, North Carolina, 27711 Tel: 919-541-1585; fax 919-541-4017 e-mail: zucker.robert@epa.gov .
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