Camila, Cells that have compromised plasma membranes will be positive for annexin. In those cells, internal PS will bind annexin, and the PI will show them to be dead. Interpretation becomes a question of seeing apoptosis as a progressive process. Unaffected cells should be annexin-/PI-, early apoptotic (where PS has flipped) should be annexin+/PI-, but these will be progressing towards death and their membranes may also be slightly permeable, so some of these may start to stain with PI. Finally, as cells progress, more will stain brightly with PI, but continue to be annexin+. It has been observed that "necrotic" cells (cells dead before apoptotic induction) may show lower annexin positivity, and thus resolve as a slightly dimmer annexin+ group in the double positive region, but I think that depend more on case characteristics. MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Flow Core 7416 CCGC 0946 (734) 647-3216, fax (734) 936-7376 kukuru@umich.edu >>> Camila Rodrigues Cacere <camila@lim56.fm.usp.br> 05/24/01 10:33AM >>> 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.
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