Analysis of apoptosis by cytometry using TUNEL assay
Analysis of apoptosis by cytometry using TUNEL assay
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DOI：
10.1016/j.ymeth.2007.11.008
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发表时间：
2008-03-01
期刊：
影响因子：
4.8
通讯作者：
Zhao, Hong
中科院分区：
文献类型：
--
作者：
Darzynkiewicz, Zbigniew;Galkowski, Dariusz;Zhao, Hong
优先在核小体间部分切割染色体DNA的核酸内切酶的激活是细胞凋亡的标志。因此，通过大量DNA链断裂的存在所揭示的DNA片段化被认为是鉴定凋亡细胞的金标准。我们已经开发了几种方法的变体，该方法基于使用外源性末端脱氧核苷酸转移酶（TdT）原位荧光标记DNA链断裂的3 '-OH末端，通常定义为TUNEL测定。本章描述了基于Br-dUTP标记的链断裂的变体，随后用Br-dUAb进行免疫细胞化学检测。与其他TUNEL变体相比，Br-dU标记测定在检测DNA断裂方面提供了最高的灵敏度。还描述了该方案的修改，其允许使用Br-dUTP荧光染料标记的脱氧核苷酸以外的标记DNA断裂。用丙啶或4 '，6-二脒基-2-苯基吲哚（DAPI）同时染色DNA和通过流式细胞术或激光扫描细胞术对细胞进行多参数分析使得能够将细胞凋亡的诱导与细胞周期相关联。爱思唯尔公司出版
Activation of endonucleases that cleave chromosomal DNA preferentially at internucleosomal sections is a hallmark of apoptosis. DNA fragmentation revealed by the presence of a multitude of DNA strand breaks, therefore, is considered to be the gold standard for identification apoptotic cells. Several variants of the methodology that is based on fluorochrome-labeling of 3'-OH termini of DNA strand breaks in situ with the use of exogenous terminal deoxynucleotidyl transferase (TdT), commonly defined as the TUNEL assay, have been developed by us. This Chapter describes the variant based on strand breaks labeling with Br-dUTP that is subsequently detected immunocytochemically with Br-dUAb. Compared with other TUNEL variants the Br-dU-labeling assay offers the greatest sensitivity in detecting DNA breaks. Described also are modifications of the protocol that allow one to use other than Br-dUTP fluorochrome-tagged deoxynucleotides to label DNA breaks. Concurrent staining of DNA with propidium or 4',6-diamidino-2-phenylindole (DAPI) and multiparameter analysis of cells by flow- or laser scanning cytometry enables one to correlate induction of apoptosis with the cell cycle phase. Published by Elsevier Inc.