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{"title":"Analysis of Cellular DNA Content by Flow Cytometry","authors":"Zbigniew Darzynkiewicz, Xuan Huang, Hong Zhao","doi":"10.1002/cpcy.28","DOIUrl":null,"url":null,"abstract":"<p>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.</p>","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":"82 1","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.28","citationCount":"27","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current Protocols in Cytometry","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpcy.28","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"Health Professions","Score":null,"Total":0}
引用次数: 27
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Abstract
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.
流式细胞术分析细胞DNA含量
细胞DNA含量可以通过流式细胞术测量，其目的是:(1)揭示细胞周期主要阶段的细胞分布，(2)估计具有部分DNA含量的凋亡细胞的频率，和/或(3)揭示被测细胞群的DNA倍性。在本单元中，介绍了简单而普遍适用的固定细胞染色方法，以及利用洗涤剂和/或蛋白水解处理来渗透细胞并使荧光染料可以接触DNA的方法。此外，描述了用Hoechst 33342进行上活体细胞染色，该染色主要用于根据dna含量差异对活细胞进行分类，以便进行后续培养。还介绍了从石蜡包埋组织中分离的细胞核染色方法。列出了DNA含量-频率直方图反褶积的可用算法，以估计细胞周期主要阶段的细胞百分比和具有分数DNA含量的凋亡细胞的频率。©2017 by John Wiley &儿子,Inc。
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