Fluorescence-activated cell sorting analysis of mitochondrial content, membrane potential, and matrix oxidant burden in human lymphoblastoid cell lines.
Fluorescence-activated cell sorting analysis of mitochondrial content, membrane potential, and matrix oxidant burden in human lymphoblastoid cell lines.
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DOI：
10.1007/978-1-61779-504-6_16
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发表时间：
2012
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影响因子：
--
通讯作者：
Falk, Marni J
中科院分区：
文献类型：
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作者：
Dingley, Stephen;Chapman, Kimberly A;Falk, Marni J
荧光激活细胞分选（FACS）允许基于其独特的荧光性质以高通量方式定量细胞群体的特定生物学参数。使用FACS分析对人细胞中的线粒体定位染料进行相对定量允许对各种线粒体参数进行灵敏分析，包括线粒体含量、线粒体膜电位和基质氧化剂负荷。在这里，我们描述的协议，利用流式细胞仪分析的人淋巴母细胞系（LCL）的相对定量的尿道局部荧光染料强度。描述的特定染料包括评估线粒体含量的MitoTracker绿色FM、评估线粒体膜电位的四甲基罗丹明乙酯（TMRE）和评估线粒体基质氧化剂负荷的MitoSOX Red。基于FACS的线粒体分析的代表性结果证明了来自健康个体的LCL中这三个基本线粒体参数的可变性，以及应用LCL的FACS分析来研究药理学诱导和清除氧化应激的作用的灵敏度。
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.