{"title":"Local Generation and Imaging of Hydrogen Peroxide in Living Cells","authors":"Yulia A. Bogdanova, Carsten Schultz, Vsevolod V. Belousov","doi":"10.1002/cpch.20","DOIUrl":null,"url":null,"abstract":"<p>Described here is a localized H<sub>2</sub>O<sub>2</sub> generation-detection system consisting of a yeast <span>D</span>-amino acid oxidase (DAAO) and two spectrally distinct variants of biosensor, HyPer2 and HyPerRed based on circularly permutated yellow and red fluorescent proteins, respectively, which enables spatiotemporal production and examination of the intracellular H<sub>2</sub>O<sub>2</sub> dynamics. The protocol describes using this system in a simple cell culture model. We provide detailed instructions on imaging of H<sub>2</sub>O<sub>2</sub> generated by the activated DAAO. The system can be easily optimized for various combinations of cell types, conditions and DAAO/sensor subcellular localizations. © 2017 by John Wiley & Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"9 2","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2017-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.20","citationCount":"22","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current protocols in chemical biology","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpch.20","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 22
Abstract
Described here is a localized H2O2 generation-detection system consisting of a yeast D-amino acid oxidase (DAAO) and two spectrally distinct variants of biosensor, HyPer2 and HyPerRed based on circularly permutated yellow and red fluorescent proteins, respectively, which enables spatiotemporal production and examination of the intracellular H2O2 dynamics. The protocol describes using this system in a simple cell culture model. We provide detailed instructions on imaging of H2O2 generated by the activated DAAO. The system can be easily optimized for various combinations of cell types, conditions and DAAO/sensor subcellular localizations. © 2017 by John Wiley & Sons, Inc.
活细胞中过氧化氢的局部生成和成像
本文描述了一个局部H2O2生成检测系统，该系统由酵母d -氨基酸氧化酶(DAAO)和两个光谱上不同的生物传感器变体HyPer2和HyPerRed组成，分别基于循环排列的黄色和红色荧光蛋白，可以实现细胞内H2O2动态的时空产生和检测。该协议描述了在一个简单的细胞培养模型中使用该系统。我们提供了活化DAAO产生的H2O2成像的详细说明。该系统可以很容易地针对细胞类型、条件和DAAO/传感器亚细胞定位的各种组合进行优化。©2017 by John Wiley &儿子,Inc。
本文章由计算机程序翻译，如有差异，请以英文原文为准。
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