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 cytotoxicity rely on radioactive tracers, which either detect the release of cytoplasmic contents after plasma membrane disintegration by dying cells (51Cr release), or retained DNA by living cells (the JAM test). In this study, the annexin V binding assay of early apoptosis was applied 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 and the results compared with those obtained by the JAM test. Assaying annexin V binding by indirect immunofluorescence was demonstrated to be more sensitive and faster than the JAM test, which is a well-described, sensitive and simple assay for DNA fragmentation and cell death. However, the annexin V binding method was considered a more accurate measurement of absolute cytotoxicity as individual cell lysis was detected directly. In other methods, cytotoxic activity was calculated indirectly as a percentage of retained or released radioactive label. In addition, the apoptosis induced by the cell-mediated cytotoxicity can be visualized by this method thereby allowing a more accurate and sensitive quantitation of the number of apoptotic cells present when low effector to target ratios are used. These advantages make the annexin V binding method superior to other conventional cytotoxicity assays, particularly in situations where effector cells can be easily distinguished or separated from target cells.