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{"title":"Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining","authors":"Paolo Cappella, Fabio Gasparri, Maurizio Pulici, Jürgen Moll","doi":"10.1002/0471142956.cy0734s72","DOIUrl":null,"url":null,"abstract":"<p>Determination of incorporation of the thymidine analog 5-bromo-2′-deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle. However, DNA denaturation is required for BrdU detection with the consequence that most protein epitopes are destroyed and their immunocytochemical detection for multiplex analysis is not possible. A novel assay is presented for identifying cells in active S-phase that does not require the DNA denaturation step but nevertheless detects BrdU. For this purpose, cells were pulsed for a short time by 5-ethynyl-2′-deoxyuridine (EdU) which is incorporated into DNA. The nucleotide-exposed ethynyl residue was then derivatized by a copper-catalyzed cycloaddition reaction (“click chemistry” coupling) using a BrdU azide probe. The resulting DNA-bound bromouracil moieties were then detected by commercial anti-BrdU monoclonal antibodies without the need for a denaturation step. This method has been tested using several cell lines and is more sensitive than traditional BrdU and allows multicolor and multiplex analysis in flow cytometry (FCM) and image-based cytometry. © 2015 by John Wiley & Sons, Inc.</p>","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2015-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/0471142956.cy0734s72","citationCount":"27","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current Protocols in Cytometry","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/0471142956.cy0734s72","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
Determination of incorporation of the thymidine analog 5-bromo-2′-deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle. However, DNA denaturation is required for BrdU detection with the consequence that most protein epitopes are destroyed and their immunocytochemical detection for multiplex analysis is not possible. A novel assay is presented for identifying cells in active S-phase that does not require the DNA denaturation step but nevertheless detects BrdU. For this purpose, cells were pulsed for a short time by 5-ethynyl-2′-deoxyuridine (EdU) which is incorporated into DNA. The nucleotide-exposed ethynyl residue was then derivatized by a copper-catalyzed cycloaddition reaction (“click chemistry” coupling) using a BrdU azide probe. The resulting DNA-bound bromouracil moieties were then detected by commercial anti-BrdU monoclonal antibodies without the need for a denaturation step. This method has been tested using several cell lines and is more sensitive than traditional BrdU and allows multicolor and multiplex analysis in flow cytometry (FCM) and image-based cytometry. © 2015 by John Wiley & Sons, Inc.
细胞增殖方法:基于BrdU偶联多重抗体染色的点击化学
测定胸腺嘧啶类似物5-溴-2 ' -脱氧尿嘧啶(BrdU)与DNA的结合是一种广泛使用的分析细胞周期的方法。然而,BrdU检测需要DNA变性,其结果是大多数蛋白质表位被破坏,无法进行多重分析的免疫细胞化学检测。提出了一种新的鉴定活性s期细胞的方法,该方法不需要DNA变性步骤,但仍然可以检测到BrdU。为此,细胞被合并到DNA中的5-乙基-2 ' -脱氧尿苷(EdU)短时间脉冲。然后使用BrdU叠氮化物探针通过铜催化的环加成反应(“点击化学”偶联)衍生化暴露于核苷酸的乙基残基。然后用商用抗brdu单克隆抗体检测得到的dna结合的溴酸基团,而不需要变性步骤。该方法已在多个细胞系中进行了测试,比传统的BrdU更敏感,并允许在流式细胞术(FCM)和基于图像的细胞术中进行多色和多路分析。©2015 by John Wiley &儿子,Inc。
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