Hello Ger... A significant problem with detecting apoptosis in adherent cell lines is cell damage associated with removal from their substrate - scraping or trypsinization can alter annexin V binding and membrane permeability, muddying up your cell death assay. If you have access to a laser scanning cytometer (LSC), you can do "flow" analysis on the cells in the adherent state - we find this gives a much cleaner assay. "Late" apoptotic cells will round up and release from the slide, but cells with "earlier" markers (such as annexin V binding, caspase activation, 7-AAD permeability) are still attached and can be detected. In our hands, serum removal induces cell cycle arrest but not apoptosis (at least not soon enough!) in murine fibroblasts. A protein transcription or translation inhibitor (such as actinomycin D or cycloheximide) induces apoptosis in these cells and may make a useful positive control - cycloheximide at 10 ug/ml within 4 hours, actinomycin D at 5 ug/ml within 12 hours. Regards, Bill Telford NCI-NIH At 11:29 AM 2/12/2002 +0100, Gerard Bowe wrote: >Hello everyone, > >We are looking for a reliable apoptotic assay for Balb/c 3T3 >(fibroblasts). We have tried the Annexin V FITC/PI with little success. >After inducing apoptosis by serum removal we get PI +ve, but FITC -ve. >We've tried various methods of inducing apoposis, but got little or no >FITC +ve. We detach the cells using trypsin. > >Any suggestions/ help would be appreciated. > > >Thanking you, >Ger
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