Hi Camila, On a dual plot of annexin (x axis) v PI (y axis), your live cells will be in the bottom left corner. Early apoptotic cells will be in the lower right quadrant ie annexin+ but PI-. As cells become more permeable to PI they will appear in the upper right quadrant ie will be Annexin+ and PI+. These cells will be dead ie late apoptotic and necrotic. Later on if they lose cytoplasm, shrink, generally blow up there may be a population that is annexin- but PI+. A quick look down the fluorescence microscope will help identifying these. You have to remember that in all live cells assays like this, we are looking at a snapshot of a dynmamic process. If you have induced apoptosis cells will be cointinually moving in an anticlockwise loop on that dual parameter plot starting in the lower left quadrant. Also you may find that some cell types give a nice clear annexin only population whereas others will have an 'apoptotic' population that is also slightly PI positive presumably due to slight permeability cahnges in early apoptosis. Derek On Thu, 24 May 2001, Camila Rodrigues Cacere wrote: > We have been studying the levels of apoptosis in peripheral blood > mononuclear cells of patients with paracoccidioidomycosis. These cells > are cultivated for 4-7 days with a specific fungal antigen, and we are > assessing the apoptosis through Annexin-V and PI. > Do someone know how to interpret those cells that are double stained > (FITC+PI): cells that are in late apotpsosis process? Necrosis? In our > cultures we detect very few cells stained with PI only, but significant > numbers of cells stained with FITC only, double stained cells, or (the > majority, live) non-stained cells. > ************************************************************************ Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk London, UK mobile: 07790 604112 Web Page: http://www.icnet.uk/axp/facs/davies/index.html In tenebris lux *************************************************************************
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