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Biochem Biophys Res Commun. 2007 Jun 22;358(1):203-8. Epub 2007 Apr 25.

Simple quantitative detection of mitochondrial superoxide production in live cells.

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Section on Oxidative Stress Tissue Injury, Laboratory of Physiological Studies, National Institutes of Health/NIAAA, Bethesda, MD 20892-9413, USA.


Experiments with isolated mitochondria have established that these organelles are pivotal intracellular sources of superoxide in a variety of pathophysiological conditions. Recently, a novel fluoroprobe MitoSOX Red was introduced for selective detection of superoxide in the mitochondria of live cells and was validated with confocal microscopy. Here we show approximately 3-7 fold dose- and time-dependent increase in mitochondrial superoxide production (measured by MitoSOX using flow cytometry and confocal microscopy) in rat cardiac derived H9c2 myocytes and/or in human coronary artery endothelial cells triggered by Antimycin A, Paraquat, Doxorubicin or high glucose. These results establish a novel, quantitative method for simple detection of mitochondrial superoxide generation simultaneously in a large population of live cells by flow cytometry. This method can also be adapted for immune cell studies with mixed population of T or B cells or their subsets to analyze mitochondrial superoxide levels using multiple labeled surface markers in individual populations.

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