Simple quantitative detection of mitochondrial superoxide production in live cells.
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.
Mukhopadhyay
Partha
P
Section on Oxidative Stress Tissue Injury, Laboratory of Physiological Studies, National Institutes of Health/NIAAA, Bethesda, MD 20892-9413, USA.
Rajesh
Mohanraj
M
Yoshihiro
Kashiwaya
K
Haskó
György
G
Pacher
Pál
P
D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology; D004317:Doxorubicin / Q000494:pharmacology; D042783:Endothelial Cells / Q000737:chemistry* / Q000187:drug effects; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D018613:Microscopy, Confocal; D008929:Mitochondria, Heart / Q000737:chemistry* / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000737:chemistry* / Q000187:drug effects; D010269:Paraquat / Q000494:pharmacology; D010617:Phenanthridines; D051381:Rats; D013481:Superoxides / Q000032:analysis*
C521281:5-(6'-triphenylphosphoniumhexyl)-5,6-dihydro-6-phenyl-3,8-phenanthridinediammine; D005456:Fluorescent Dyes; D010617:Phenanthridines; D013481:Superoxides; D000968:Antimycin A; D004317:Doxorubicin; D005947:Glucose; D010269:Paraquat