Hello Paul (and everyone)... It is probably correct to not consider annexin V to be an "early" apoptotic event anymore, at least relative to other detectable indicators of cell death. In most of our apoptosis systems (i.e. such as mouse thymoma cells undergoing apoptosis either due to media deprivation or drug treatment), PS flipping is preceded by DNA fragmentation (via TUNEL), mitochondrial membrane potential flux (via DioC6(3) or JC-1), caspase 3 activation (by PhiPhiLux or by anti-active caspase 3 antibody detection) and by early cell volume fluctuations. 7-AAD permeability occurs roughly simultaneous to annexin V binding - PI permeability, cytoskeletal collapse and surface detachment seem to occur later. We get rather convincing annexin V(+) PI (-) subpopulations that we take to be "intermediate" apoptotic cells - however, since annexin V binding occurs rather close in time to loss of membrane permeability, it might make one wonder whether it is getting inside the cell at this point. Once again, doing multiple assays for cell death rather than relying on one characteristic seems to be the best option. Bill At 09:27 AM 8/3/01 +1000, Paul Waring wrote: > >I notice a brief flurry of questions on annexinV staining and whether it is >"early" or "late". We showed (Cell Death and Diff. 1999, vol6, 624) that it >precedes (only just) DNA fragmentation in thymocytes treated with three >different agents to induce apoptosis with different kinetics (thapsigargin, >gliotoxin and dexamethasone). I would not rate this as an "early" event in >apoptosis. We also report in the same paper that necrosis can lead to >PS(+)PI(-) cells (I'm talking a large population - 32%). In fact the FACS >profile at 1 hr when treated with the necrosis inducer was the same as the >FACS profile at 6 hrs of the same cells treated with dex! > >I would not use Annexin V staining (even with PI) as a primary assay for >apoptosis > >hope this is some help > >Paul Waring > >NHMRC Senior Research Fellow >Chemistry, The faculties >ANU, Australia.
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