Supravital exposure to propidium iodide identifies apoptosis on adherent cells

Background: Several studies indicate that plasma membrane changes during ap optosis are a general phenomenon. Among the flow cytometric methods to meas ure apoptosis, the Annexin V assay that detects the membrane exposure of ph osphatidylserine (PS) is one of the most commonly used. However, the variou s treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods: Apoptosis of different cell, lines was investigated by fluorescence microscopy and multiple now assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light sc atter changes. Results and Conclusions: We show that supravital propidium iodide (PT) assa y stains adherent apoptotic cells, allowing flow cytometric quantification. Moreover, supravital exposure to PI without prior permeabilization identif ies apoptotic cells as well as Annexin V and permits the simultaneous surfa ce staining by FITC- and PE-conjugated monoclonal antibodies. As in the cas e of necrotic or permeabilized cells, fluorescence microscopy has revealed that PI staining of apoptotic cells is localized in the nucleus. This sugge sts that the binding of PI to the DNA/ RNA structures is stable enough to w ithstand the trypsinization and/or washing procedures necessary to detach a dherent cells. Cytometry 44:57-64, 2001. (C) 2001 Wiley-Liss, Inc.