Crowley Lisa C, Marfell Brooke J, Waterhouse Nigel J
Apoptosis and Cytotoxicity Laboratory, Mater Research, Translational Research Institute, Woolloongabba, Brisbane, Queensland 4102, Australia.
Apoptosis and Cytotoxicity Laboratory, Mater Research, Translational Research Institute, Woolloongabba, Brisbane, Queensland 4102, Australia; Flow Cytometry and Imaging, QIMR Berghofer Medical Research Institute, Herston, Brisbane, Queensland 4006, Australia; School of Medicine, University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia.
Cold Spring Harb Protoc. 2016 Oct 3;2016(10):2016/10/pdb.prot087221. doi: 10.1101/pdb.prot087221.
Degradation of DNA into oligonucleosomal-sized fragments is a unique event in apoptosis that is orchestrated by caspase-activated DNase. Traditionally, this event is observed by resolving cellular DNA by gel electrophoresis, which results in a characteristic "ladder" pattern. However, this technique is time-consuming and cannot be used to quantitate the number of apoptotic cells in a sample. Terminal dUTP nick-end labeling (TUNEL) of fragmented DNA allows researchers to identify DNA fragmentation at the single-cell level. This method involves the specific addition of fluorescently labeled UTP to the 3'-end of the DNA fragments by terminal deoxynucleotidyl transferase. The TUNEL assay is both fast and sensitive. Here, we describe a protocol in which cells are treated with TUNEL reagent and counterstained with Hoechst 33342. In contrast to TUNEL, which only stains apoptotic cells, Hoechst 33342 stains the DNA of all cells.
DNA降解为寡核小体大小的片段是细胞凋亡中的一个独特事件，由半胱天冬酶激活的脱氧核糖核酸酶精心调控。传统上，通过凝胶电泳分离细胞DNA来观察这一事件，这会产生特征性的“梯状”模式。然而，该技术耗时且不能用于定量样品中凋亡细胞的数量。对断裂DNA进行末端脱氧尿苷三磷酸缺口末端标记（TUNEL）可使研究人员在单细胞水平上鉴定DNA片段化。该方法包括通过末端脱氧核苷酸转移酶将荧光标记的尿苷三磷酸特异性添加到DNA片段的3'末端。TUNEL检测既快速又灵敏。在此，我们描述一种方案，其中细胞用TUNEL试剂处理并用Hoechst 33342复染。与仅对凋亡细胞染色的TUNEL不同，Hoechst 33342对所有细胞的DNA进行染色。
