APOPTOSIS DETECTION BY ANNEXIN-V BINDING - A NOVEL METHOD FOR THE QUANTITATION OF CELL-MEDIATED CYTOTOXICITY

Current standard methods for the measurement of cell-mediated cytotoxi city rely on radioactive tracers, which either detect the release of c ytoplasmic contents after plasma membrane disintegration by dying cell s (Cr-51 release), or retained DNA by livings cells (the JAM test). In this study, the annexin V binding assay of early apoptosis was applie d to measure cell-mediated cytotoxicity. Primed human lymphocytes were examined for their ability to lyse either xenogeneic pig endothelial or allogeneic human PBMC target cells by assaying annexin V binding an d the results compared with those obtained by the JAM test. Assaying a nnexin V binding by indirect immunofluorescence was demonstrated to be more sensitive and faster than the JAM test, which is a well-describe d, sensitive and simple assay for DNA fragmentation and cell death. Ho wever, the annexin V binding method was considered a more accurate mea surement of absolute cytotoxicity as individual cell lysis was detecte d directly. In other methods, cytotoxic activity was calculated indire ctly as a percentage of retained or released radioactive label. In add ition, the apoptosis induced by the cell-mediated cytotoxicity can be visualized by this method thereby allowing a more accurate and sensiti ve quantitation of the number of apoptotic cells present when low effe ctor to target ratios are used. These advantages make the annexin V bi nding method superior to other conventional cytotoxicity assays, parti cularly in situations where effector cells can be easily distinguished or separated from target cells. (C) 1998 Elsevier Science B.V. All ri ghts reserved.