Darzynkiewicz Zbigniew, Huang Xuan, Zhao Hong
Department of Pathology and Brander Cancer Research Institute, New York Medical College, Valhalla, New York.
Hematology/Oncology, Case Western Reserve University Cleveland, Ohio.
Curr Protoc Immunol. 2017 Nov 1;119:5.7.1-5.7.20. doi: 10.1002/cpim.36.
Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA-content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin-embedded tissues. Available algorithms are listed for deconvolution of DNA-content-frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content. © 2017 by John Wiley & Sons, Inc.
细胞DNA含量可通过流式细胞术进行测量，目的如下：(1) 揭示细胞在细胞周期主要阶段的分布情况；(2) 利用DNA含量分数估计凋亡细胞的频率；和/或(3) 揭示被测细胞群体的DNA倍性。在本单元中，介绍了用于固定细胞染色的简单且普遍适用的方法，以及利用去污剂和/或蛋白水解处理使细胞通透并使DNA能够被荧光染料染色的方法。此外，还描述了用Hoechst 33342进行的超活细胞染色，该方法主要用于根据DNA含量差异对活细胞进行分选以便后续培养。还介绍了从石蜡包埋组织中分离细胞核的染色方法。列出了可用的算法，用于对DNA含量频率直方图进行反卷积，以估计细胞周期主要阶段的细胞百分比和具有分数DNA含量的凋亡细胞频率。© John Wiley & Sons, Inc. 2017年版权所有。
