Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy

被引：308

作者：

Mukhopadhyay, Partha ^{[1
]}

Rajesh, Mohanraj ^{[1
]}

Hasko, Gyoergy ^{[2
]}

Hawkins, Brian J. ^{[3
]}

Madesh, Muniswamy ^{[3
]}

Pacher, Pal ^{[1
]}

机构：

[1] NIAAA, Sect Oxidat Stress Tissue Injury, Lab Physiol Studies, NIH, Bethesda, MD 20892 USA

[2] Univ Med & Dent New Jersey, New Jersey Med Sch, Dept Surg, Newark, NJ 07103 USA

[3] Univ Penn, Inst Environm Med, Dept Canc Biol, Philadelphia, PA 19104 USA

来源：

NATURE PROTOCOLS | 2007年 / 2卷 / 09期

关键词：

D O I：

10.1038/nprot.2007.327

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Annexin V and Sytox Green are widely used markers to evaluate apoptosis in various cell types using flow cytometry and fluorescent microscopy. Recently, a novel fluoroprobe MitoSOX Red was introduced for selective detection of superoxide in the mitochondria of live cells and was validated for confocal microscopy and flow cytometry. This protocol describes simultaneous measurements of mitochondrial superoxide generation with apoptotic markers (Annexin V and Sytox Green) by both flow cytometry and confocal microscopy in endothelial cell lines. The advantages of the described flow cytometry method over other cell-based techniques are the tremendous speed (1-2 h), exquisite precision and the possibility of simultaneous quantitative measurements of mitochondrial superoxide generation and apoptotic (and other) markers, with maximal preservation of cellular functions. This method combined with fluorescent microscopy may be very useful to reveal important spatial-temporal changes in mitochondrial superoxide production and execution of programmed cell death in virtually any cell type.

引用

页码：2295 / 2301

页数：7

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