Donner K J, Becker K M, Hissong B D, Ahmed S A
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.
Cytometry. 1999 Jan 1;35(1):80-90. doi: 10.1002/(sici)1097-0320(19990101)35:1<80::aid-cyto11>3.3.co;2-#.
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.
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.
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.
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.
测量细胞凋亡的技术被用于研究从获得性免疫缺陷综合征（艾滋病）到癌症再到自身免疫性疾病等广泛的病症。因此，对常见细胞凋亡检测方法进行关键比较是很有必要的。
在培养2、4、8、12、26 - 28和48 - 50小时后，检测地塞米松（DEX）处理的胸腺细胞中细胞凋亡诱导的动力学。另一个目的是确定一种类似的诱导胸腺萎缩的激素己烯雌酚（DES）是否也直接诱导胸腺细胞凋亡。通过对用碘化丙啶（PI）、7 - 氨基放线菌素D（7 - AAD）或异硫氰酸荧光素（FITC） - 膜联蛋白染色的细胞进行流式细胞术检测来评估细胞凋亡；通过前向和侧向散射（FS、SS）分析、细胞大小分析仪以及通过细胞病理学检查来评估。
DEX处理4小时后，7 - AAD、膜联蛋白和细胞病理学检测显示细胞凋亡明显，但PI分析未发现具有亚二倍体DNA含量的细胞。DEX处理12小时后，上述所有流式细胞术技术均显示最大程度的细胞凋亡。7 - AAD和膜联蛋白检测能够区分早期凋亡和晚期凋亡/坏死，二者相当且识别出相似的凋亡群体。仅在DEX处理12小时后才明显出现FSlow/SS增加的群体。在暴露于不同剂量DES的胸腺细胞中，上述任何检测方法均未检测到细胞凋亡。
六种检测方法中的两种，即7 - AAD和膜联蛋白，在早期动力学时间点检测细胞凋亡方面相似。在所有研究的时间点，两种检测方法的结果相当。我们的研究表明，DEX和DES在体内通过不同机制诱导胸腺萎缩。
