Comparison of multiple assays for kinetic detection of apoptosis in thymocytes exposed to dexamethasone or diethylstilbesterol

Background: Techniques to measure apoptosis are used to study a wide spectr um of conditions, from acquired immune deficiency syndrome (IUDS) to cancer to autoimmune diseases. Therefore, a critical comparison of common assays for apoptosis is warranted. Methods: The kinetics of apoptosis induction in dexamethasone (DEX)-exposed thymocytes was examined after 2, 4, 8, 12, 26-28, and 48-50 h of culture. An additional aim was to ascertain whether a similar thymic atrophy-inducin g hormone, diethylstilbestrol (DES), also directly induces thymocyte apopto sis. Apoptosis was evaluated by flow cytometric examination of cells staine d with propidium iodide CPI), 7-aminoactinomycin D (7-AAD), or fluorescein isothiocyante (FITC)-annexin; by forward- and side-scatter (FS, SS) analysi s, cell-size analyzer; and through cytopathologic examination. Results: After 4 h of DEX exposure, apoptosis was evident by 7-AAD, annexin , and cytopathological assays, but no cells with sub-diploid DNA content we re evident by PI analysis. Maximal apoptosis was evident by all the above f low cytometric techniques at 12 h after DEX exposure. The 7-AAD and annexin assays, which allow discrimination between early apoptosis and late apopto sis/necrosis, were comparable and identified similar apoptotic populations. Appearance of a FSlow/SSincreased population was evident only after 12 h o f DEX exposure. Apoptosis could not be detected by any of the above assays in thymocytes exposed to various doses of DES. Conclusion: Two of the six assays, 7-AAD and annexin, were similar in detec ting apoptosis at an early kinetic time point. Results of both assays were comparable at all time points studied. Our studies imply that DEX and DES i nduce thymic atrophy, in vivo, by different mechanisms. Cytometry 35:80-90, 1999. (C) 1999 Wiley-Liss, Inc.