The selective detection of mitochondrial superoxide by live cell imaging

A general protocol is described to improve the specificity for imaging superoxide formation in live cells via fluorescence microscopy with either hydroethidine (HE) or its mitochondrially targeted derivative Mito-HE (MitoSOX Red). Two different excitation wavelengths are used to distinguish the superoxide-dependent hydroxylation of Mito-HE (385–405 nm) from the nonspecific formation of ethidium (480–520 nm). Furthermore, the dual wavelength imaging in live cells can be combined with immunocolocalization, which allows superoxide formation to be compared simultaneously in cocultures of two types of genetically manipulated cells in the same microscopic field. The combination of these approaches can greatly improve the specificity for imaging superoxide formation in cultured cells and tissues.

[1]  Elisa Nemes,et al.  Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry , 2007, Nature Protocols.

[2]  Hongtao Zhao,et al.  Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[3]  Michael S Janes,et al.  Selective fluorescent imaging of superoxide in vivo using ethidium-based probes , 2006, Proceedings of the National Academy of Sciences.

[4]  M. Madesh,et al.  Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy , 2007, Nature Protocols.

[5]  T. Hurd,et al.  Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology , 2005, Biochemistry (Moscow).

[6]  S. Flanagan,et al.  Mutant SOD1‐induced neuronal toxicity is mediated by increased mitochondrial superoxide levels , 2007, Journal of neurochemistry.

[7]  M. Brand,et al.  Prevention of mitochondrial oxidative damage as a therapeutic strategy in diabetes. , 2004, Diabetes.

[8]  B. Kalyanaraman,et al.  Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: identification of homo- and heterodimers. , 2008, Free radical biology & medicine.

[9]  Hongtao Zhao,et al.  Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. , 2003, Free radical biology & medicine.

[10]  L. Barbeito,et al.  Mitochondrial Superoxide Production and Nuclear Factor Erythroid 2-Related Factor 2 Activation in p75 Neurotrophin Receptor-Induced Motor Neuron Apoptosis , 2007, The Journal of Neuroscience.

[11]  B. Kalyanaraman,et al.  The confounding effects of light, sonication, and Mn(III)TBAP on quantitation of superoxide using hydroethidine. , 2006, Free radical biology & medicine.

[12]  E. Banfi,et al.  A new flow cytometric assay for the evaluation of phagocytosis and the oxidative burst in whole blood. , 1994, Journal of immunological methods.

[13]  Gregor Rothe,et al.  Flow Cytometric Analysis of Respiratory Burst Activity in Phagocytes With Hydroethidine and 2′,7′‐Dichlorofluorescin , 1990, Journal of leukocyte biology.

[14]  L. Barbeito,et al.  Peroxynitrite triggers a phenotypic transformation in spinal cord astrocytes that induces motor neuron apoptosis , 2002, Journal of neuroscience research.

[15]  B. Ames,et al.  Oxidative damage and mitochondrial decay in aging. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[16]  K. Tipton,et al.  Uptake and accumulation of 1-methyl-4-phenylpyridinium by rat liver mitochondria measured using an ion-selective electrode. , 1992, The Biochemical journal.