Y. Shounan et al., APOPTOSIS DETECTION BY ANNEXIN-V BINDING - A NOVEL METHOD FOR THE QUANTITATION OF CELL-MEDIATED CYTOTOXICITY, Journal of immunological methods, 217(1-2), 1998, pp. 61-70
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.