Jose Benito wrote: > > I want to study apoptosis in PBMCs using the staining with annexin as a > marker of apoptotic cells. I have some questions regarding this issue: > 1.- How good the annexin staining correlates with apoptosis. In a PBMC system, we found annexin to correlate well with other assays of apoptosis. Though the median % apoptosis determined with annexin was the highest, it was not significantly different from the other assays. for more details see: AIDS Res Hum Retro, 14: 1413-1422, 1998. Can a cell be > defined as apoptoic simply based in the annexin criterion? I would not define a cell as apoptotic based on one assay. It is best to use a couple of assays and essential to examine nuclear morphology by fluorescence cmicroscopuy to confirm apoptosis. > 2.- Is there any test rally specific for the fenomenon of apoptosis? Cellular morphology is still the hallmark of this process, for flow assays we have found TUNEL to prerform very well. > 3.- What is the rationale of using propidium iodide in combination with > annexin? Is it to differentiate apoptotic from necrotic cells? If this is > the case, does it mean that cells positive for annexin and negative for PI > should be classified as apoptotic and double positive cells as necrotic? PI assesses membrane integrity, which is disrupted in necrotic or late apoptotic cells. In an assay system in which all cells are dying via apoptotic mechanisms and you wish to quantify total apoptosis, I';m not sure it adds much info. Good luck, Tom ***************************************************************************** Thomas W. Mc Closkey, Ph. D. Director of Flow Cytometry, North Shore University Hospital Assistant Professor of Pediatrics, New York University School of Medicine Boas Marks Biomedical Research Center, 350 Community Drive Manhasset, Long Island, New York 11030 ph: 516-562-4844 [office], 516-562-1135/4641 [lab] fax: 516-562-2866 *****************************************************************************
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