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

Abstract

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. Conclusion: 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. Cytometry 35:80–90, 1999. © 1999 Wiley‐Liss, Inc.

Tactivin in the regulation of dexamethasone-induced apoptosis in thymocytes

“…2, b, c). It is known that DM triggers cascade reactions of cell apoptosis; its effect develops after 12 h [9] in vivo and according to our data not less than after 6 h in vitro.…”

Malathion, Lindane, and Piperonyl Butoxide, Individually or in Combined Mixtures, Induce Immunotoxicity via Apoptosis in Murine Splenocytes In Vitro

“…Slides were allowed to dry and were stained with modified Wright stain (Sigma). 30,31 Cover slips were adhered to the slides using Permount (Fisher Scientific, Pittsburgh, Pa). After 24 hours, slides were examined under oil immersion light microscopy (Â250) on the Olympus AH-2 Vanox-T Light Microscope.…”

Serine protease inhibitor, 4-(2-aminoethyl)-benzene sulfonyl fluoride, impairs IL-12-induced activation of pSTAT4β, NFκB, and select pro-inflammatory mediators from estrogen-treated mice

“…The percentage of viable lymphocytes or stages of cell death was determined by 7-AAD as described in previous studies (Donner et al 1999). Staining with 7-AAD has been shown to be reliable and sensitive in identifying apoptotic lymphocytes, particularly in early stages of apoptosis (Donner et al 1999).…”

“…Staining with 7-AAD has been shown to be reliable and sensitive in identifying apoptotic lymphocytes, particularly in early stages of apoptosis (Donner et al 1999). Briefly, 100 l of splenic lymphocytes (5 × 10 6 cells/ml) were cultured with equal volumes of ConA (10 g/ml) or rIL-12 (20 ng/ml) for 3 h. Select cultures received AEBSF.…”