哺乳动物细胞和酵母中线粒体氧化还原状态的活细胞成像。
Live cell imaging of mitochondrial redox state in mammalian cells and yeast.
作者信息
Liao Pin-Chao, Franco-Iborra Sandra, Yang Yi, Pon Liza A
机构信息
Department of Pathology and Cell Biology, Columbia University, New York, NY, United States.
Synthetic Biology and Biotechnology Laboratory, Key State Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai, China.
出版信息
Methods Cell Biol. 2020;155:295-319. doi: 10.1016/bs.mcb.2019.11.008. Epub 2020 Feb 14.
Abstract
The redox state of mitochondria is determined by the levels of reducing and oxidizing species in the organelle, which reflects mitochondrial metabolic activity and overall fitness. Mitochondria are also the primary endogenous source of reactive oxygen species (ROS). This chapter describes methods to measure the mitochondrial superoxide levels and the redox state of the organelle in mammalian cells and yeast. We describe the use of dihydroethidium (DHE) and MitoSOX (a derivative of dihydroethidium bound to a lipophilic cation) to detect mitochondrial superoxide in yeast and mammalian cells, respectively. We also describe the use of genetically encoded fluorescent biosensors for quantitative analysis of mitochondrial NADPH levels (iNap) in mammalian cells and mitochondrial redox state (mito-roGFP) in yeast.
摘要
线粒体的氧化还原状态由细胞器中还原型和氧化型物质的水平决定，这反映了线粒体的代谢活性和整体健康状况。线粒体也是活性氧（ROS）的主要内源性来源。本章介绍了测量哺乳动物细胞和酵母中线粒体超氧化物水平以及细胞器氧化还原状态的方法。我们描述了分别使用二氢乙锭（DHE）和MitoSOX（与亲脂性阳离子结合的二氢乙锭衍生物）来检测酵母和哺乳动物细胞中的线粒体超氧化物。我们还描述了使用基因编码的荧光生物传感器对哺乳动物细胞中线粒体NADPH水平（iNap）和酵母中线粒体氧化还原状态（mito-roGFP）进行定量分析的方法。
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