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The selective detection of mitochondrial superoxide by live cell imaging

Nature Protocols volume 3, pages 941947 (2008) | Download Citation



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

    • Kristine M Robinson
    • , Michael S Janes
    •  & Joseph S Beckman
  2. Invitrogen–Molecular Probes Labeling and Detection Technologies, Eugene, Oregon 97402, USA.

    • Michael S Janes


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Competing interests

Mike Janes is an employee of Invitrogen, Inc.

Corresponding author

Correspondence to Joseph S Beckman.

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