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The selective detection of mitochondrial superoxide by live cell imaging

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Abstract

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.

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Figure 1: Oxidation of hydroethidine (HE) and Mito-HE.
Figure 2: Detection of superoxide production from two wavelengths in an astrocyte expressing the amyotrophic lateral sclerosis-associated mutation SODG93A to copper, zinc superoxide dismutase.
Figure 3: The detection of increased superoxide production in SODG93A astrocytes cocultured with nontransgenic astrocytes.

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Linus Pauling Institute, Environmental Health Sciences Center, Oregon State University, Corvallis, 97331, Oregon, USA

    Kristine M Robinson, Michael S Janes & Joseph S Beckman

  2. Invitrogen–Molecular Probes Labeling and Detection Technologies, Eugene, 97402, Oregon, USA

    Michael S Janes

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  1. Kristine M Robinson
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  2. Michael S Janes
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  3. Joseph S Beckman
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Correspondence to Joseph S Beckman.

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Mike Janes is an employee of Invitrogen, Inc.

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Robinson, K., Janes, M. & Beckman, J. The selective detection of mitochondrial superoxide by live cell imaging. Nat Protoc 3, 941–947 (2008). https://doi.org/10.1038/nprot.2008.56

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