I've been following the discussions on AnnexinV and decided to put in my two cents. A concept that a lot of people seem to miss is that AnnexinV is more than useless when used alone, it is misleading. You have to use it in conjunction with a dead cell discriminator. Dead cells stain very brightly for Annexin V. And no, AnnexinV+/PI+ double positives do not imply late apoptosis or apoptotic death. The PI is only used so that you don't count those cells as apoptotic. They are simply dead and that is all you can say about them. Also, timing is everything. AnnexinV is only useful for EARLY apoptosis. You may have to do some experimentation to determine the proper time point for measurement. If you find you are really interested in late apoptosis, use a tunel kit Sometimes you must come to the realization that AnnexinV is not the best apoptosis assay for your system. I find, as someone else pointed out, that adherent cells tend to give a high percent of background. Physical perturbation of the cells can cause PS flipping and false positives. Controls are paramount. You need to have a non-apoptotic control that is indeed AnnexinV negative, or at least dim. You also need to have a positive apoptotic control that is indeed Annexin V positive. If you can't get AnnexinV positivity with a strong apoptotic stimulus and Annexin V negativity with no apoptotic stimulus, how can you trust your results? Your actual test samples may be somewhere in between and you will have to decide how to interpret them based on your control. It is not always black and white. And finally, try to correlate your results with another assay. (Caspase 3 is another early apoptosis method.) I find that too often experimenters doing flow neglect the microscope. An easy fluorescence microscopy technique uses ethidium bromide and acridine orange to distinguish viable, early apoptotic, late apoptotic and necrotic cells. (Sorry I don't have the reference at my fingertips). I will reiterate what I said before, one apoptosis assay is not going to work under all experimental conditions. You may actually have to try a few things and see what works for you. And now, just to be Devil's advocate, check these out: Annexin V Binds to Positively Selected B Cells. Stacey R. Dillon, et al. J. Immun. 2001, 166: 58-71. Annexin V Binds to Viable B Cells and Colocalizes with a Marker of Lipid Rafts upon B Cell Receptor Activaiton. Dillon, et al. J. Immun. 2000, 164: 1322-1332. David McFarland GlaxoSmithKline
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