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 spectrum of conditions, from acquired immune deficiency syndrome (AIDS) 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-inducing hormone, diethylstilbestrol (DES), also directly induces thymocyte apoptosis. Apoptosis was evaluated by flow cytometric examination of cells stained with propidium iodide (PI), 7-aminoactinomycin D (7-AAD), or fluorescein isothiocyante (FITC)-annexin; by forward-and side-scatter (FS, SS) analysis, 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 were evident by PI analysis. Maximal apoptosis was evident by all the above flow cytometric techniques at 12 h after DEX exposure. The 7-AAD and annexin assays, which allow discrimination between early apoptosis and late apoptosis/necrosis, were comparable and identified similar apoptotic populations. Appearance of a FSlow/SSincreased population was evident only after 12 h of DEX exposure. Apoptosis could not be detected by any of the above assays in thymocytes exposed to various doses of DES.


CONCLUSIONS
Two of the six assays, 7-AAD and annexin, were similar in detecting 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 induce thymic atrophy, in vivo, by different mechanisms.
Donner
K J
KJ
Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg 24061, USA.
Becker
K M
KM
Hissong
B D
BD
Ahmed
S A
SA
D000818:Animals; D017304:Annexin A5 / Q000032:analysis; D017209:Apoptosis; D002273:Carcinogens / Q000494:pharmacology; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects; D004247:DNA / Q000032:analysis*; D003609:Dactinomycin / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology*; D004054:Diethylstilbestrol / Q000494:pharmacology*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D005938:Glucocorticoids / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D013950:Thymus Gland / Q000166:cytology* / Q000187:drug effects*
D017304:Annexin A5; D002273:Carcinogens; D005456:Fluorescent Dyes; D005938:Glucocorticoids; D003609:Dactinomycin; C025942:7-aminoactinomycin D; C033234:actinomycin D2; D004054:Diethylstilbestrol; D003907:Dexamethasone; D004247:DNA