Dear Lindsay, When you detach adherent cells you usually increase background positives for Annexin V. Thus, you should perform the assay with your cells still attached and then assess your results using a confocal micrsocope or a fluorescence microplate reader. It is advisable to double stain your cells with either Hoechst or PI in addition to Annexin V. In my opinion, if you insist on assessing your results by flow cytometry, you should first complete the Annexin V procedure and only then detach your cells as gently as possible. Never, never subtract calcium from your medium. Fas is by no means the only way to induce apoptosis. Unless your protocol requires it you should try inducing apoptosis by any one of dozens of other ways, like cell cycle arrest, to cite just one example. By the way, what is your usual method for inducing apoptosis? Good luck! M. Kushnir, PhD Playfair Neuroscience Unit Toronto Western Hospital Toronto, ON. M5T 2S8 Canada.
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