This has been discussed before. When one looks at apoptosis by Annexin V one often sees a pattern of Annexin V+ PI- cells early and then a pattern develops where there is a creeping up into the double positive quadrant- a smear that extends from the Annexin V+ PI-. We all know that these cells are late apoptotic cells. Although one needs to see a population of Annexin V+ PI- cells to conclude there is apoptosis, if one sets the analysis quadrants and cranks out numbers for this population only, they are inaccurate. Maryalice >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 Maryalice Stetler-Stevenson Director Flow Cytometry Unit Laboratory of Pathology, NCI, NIH "Learn the rules so you know how to break them properly." The Dalai Lama
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