A FLOW-CYTOMETRIC METHOD FOR THE SEPARATION AND QUANTITATION OF NORMAL AND APOPTOTIC THYMOCYTES
A FLOW-CYTOMETRIC METHOD FOR THE SEPARATION AND QUANTITATION OF NORMAL AND APOPTOTIC THYMOCYTES
复制标题
DOI：
10.1016/0003-2697(92)90251-2
复制
发表时间：
1992-08-01
影响因子：
2.9
通讯作者：
COHEN, GM
中科院分区：
文献类型：
--
作者：
SUN, XM;SNOWDEN, RT;COHEN, GM
利用流式细胞术，我们描述了一种分离和定量正常和凋亡胸腺细胞的方法。用糖皮质激素地塞米松或抗肿瘤药物依托泊苷诱导未成熟大鼠胸腺细胞凋亡。随后与活性双苯并咪唑染料Hoechst 33342和脱氧核糖核酸嵌入剂碘化丙啶孵育，使三种不同的细胞群能够通过流式细胞仪进行鉴定和分选。死亡细胞因碘化丙啶而发出红色荧光，而正常细胞和凋亡细胞因Hoechst 33342而发出蓝色荧光。凋亡细胞与正常胸腺细胞的区别在于它们的蓝色荧光强度更高，而前向光散射的减少决定了它们的体积更小。较大的细胞，低蓝色荧光，在电子显微镜下显示正常的胸腺细胞形态，通过琼脂糖凝胶电泳法检测没有任何DNA片断。相反，较小的细胞既表现出凋亡的形态特征，又表现出广泛的核小体间DNA片段化，约为180bp的倍数。用该方法观察到地塞米松诱导的细胞凋亡呈时间依赖性，地塞米松可被放线菌亚胺、放线菌素D和三羧酸金抑制。该方法应有助于胸腺细胞诱导凋亡的机制研究。
Using flow cytometry, we describe a method for separating and quantifying normal and apoptotic thymocytes. Apoptosis was induced in isolated thymocytes from immature rats by treatment with the glucocorticoid dexamethasone or the antitumor agent etoposide. Subsequent incubation with the vital bisbenzimidazole dye Hoechst 33342 and the DNA intercalating agent propidium iodide enabled three distinct populations of cells to be identified and sorted by flow cytometry. Dead cells fluoresced red due to propidium iodide whereas normal and apoptotic cells fluoresced blue due to Hoechst 33342. Apoptotic cells were distinguished from normal thymocytes both by their higher intensity of blue fluorescence and by their smaller size as determined by a reduction in forward light scatter. The larger cells, with low blue fluorescence, showed normal thymocyte morphology by electron microscopy and the absence of any DNA fragmentation as measured by agarose gel electrophoresis. In contrast, the smaller cells showed both the morphological characteristics of apoptosis and extensive internucleosomal fragmentation of DNA to multiples of approximately 180 bp. Using this method, a time-dependent induction of apoptosis by dexamethasone, which was inhibited by cycloheximide, actinomycin D, and aurin tricarboxylate, was observed. The method should facilitate mechanistic studies on the induction of apoptosis in thymocytes.