A simplified protocol for apoptosis assay by DNA content analysis.
A simplified protocol for apoptosis assay by DNA content analysis.
复制标题
通过 DNA 含量分析进行细胞凋亡测定的简化方案。
作者：
R. Wang;Yu
凋亡细胞具有特定的形态学和生化标记。已经开发了各种方法来检测基于这些标志物的细胞凋亡。其中最常见的是基因组DNA的片段化。除了用于鉴定特征性200碱基对梯状条带的电泳和末端脱氧核糖核苷酸转移酶介导的dUTP-生物素缺口末端标记外，还经常使用DNA含量分析。该技术基于这样的事实，即透化的凋亡细胞释放片段化的DNA，导致DNA含量低于活的二倍体细胞中的DNA含量。虽然被广泛使用，但我们发现DNA含量分析检测到的凋亡细胞数量往往低于其他方法检测到的凋亡细胞数量。我们已经开发了一个简化版本的流式细胞术为基础的协议，检测凋亡细胞的数量更接近其他方法检测到的，这需要一个显着减少的细胞数量。此外，这种简化的方案允许同时制备大量样品。
Apoptotic cells possess specific morphological and biochemical markers. Various methods have been developed to detect apoptosis based on these markers. One of the most common is the fragmentation of genomic DNA. In addition to electrophoresis for the identification of the characteristic 200 base pair ladders and terminal deoxyribonucleotidyltransferase-mediated dUTP-biotin nick end-labeling, DNA content analysis is often employed. This technique is based on the fact that permeablized apoptotic cells release fragmented DNA, resulting in DNA content that is less than that in live diploid cells. Although widely used, we have found that the number of apoptotic cells detected by DNA content analysis is often lower than that detected by other methods. We have developed a simplified version of the flow cytometry-based protocol that detects a number of apoptotic cells closer to that detected by other methods, and which requires a dramatically reduced number of cells. In addition, this simplified protocol allows preparation of a large number of samples at the same time.