Dingley Stephen, Chapman Kimberly A, Falk Marni J
Division of Human Genetics, Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
Methods Mol Biol. 2012;837:231-9. doi: 10.1007/978-1-61779-504-6_16.
Fluorescence-activated cell sorting (FACS) permits specific biologic parameters of cellular populations to be quantified in a high-throughput fashion based on their unique fluorescent properties. Relative quantitation of mitochondrial-localized dyes in human cells using FACS analysis allows sensitive analysis of a variety of mitochondrial parameters including mitochondrial content, mitochondrial membrane potential, and matrix oxidant burden. Here, we describe protocols that utilize FACS analysis of human lymphoblastoid cell lines (LCL) for relative quantitation of mitochondrial-localized fluorescent dye intensity. The specific dyes described include MitoTracker Green FM to assess mitochondrial content, tetramethylrhodamine ethyl ester (TMRE) to assess mitochondrial membrane potential, and MitoSOX Red to assess mitochondrial matrix oxidant burden. Representative results of FACS-based mitochondrial analyses demonstrate the variability of these three basic mitochondrial parameters in LCLs from healthy individuals, as well as the sensitivity of applying FACS analysis of LCLs to study the effects of pharmacologic induction and scavenging of oxidant stress.
荧光激活细胞分选（FACS）能够基于细胞群体独特的荧光特性，以高通量方式对其特定生物学参数进行定量分析。使用FACS分析对人细胞中线粒体定位染料进行相对定量，可对包括线粒体含量、线粒体膜电位和基质氧化应激负担在内的多种线粒体参数进行灵敏分析。在此，我们描述了利用人淋巴母细胞系（LCL）的FACS分析对线粒体定位荧光染料强度进行相对定量的实验方案。所描述的特定染料包括用于评估线粒体含量的MitoTracker Green FM、用于评估线粒体膜电位的四甲基罗丹明乙酯（TMRE）以及用于评估线粒体基质氧化应激负担的MitoSOX Red。基于FACS的线粒体分析的代表性结果表明，健康个体LCL中这三个基本线粒体参数存在变异性，同时也表明应用LCL的FACS分析来研究氧化应激的药物诱导和清除作用具有敏感性。
