Protocol | Published:

The selective detection of mitochondrial superoxide by live cell imaging

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

Subjects

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , & Oxidative damage and mitochondrial decay in aging. Proc. Natl. Acad. Sci. USA 91, 10771–10778 (1994).

  2. 2.

    , & Prevention of mitochondrial oxidative damage as a therapeutic strategy in diabetes. Diabetes 53 (suppl 1): S110–118 (2004).

  3. 3.

    & Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2′,7′-dichlorofluorescin. J. Leukoc. Biol. 47, 440–448 (1990).

  4. 4.

    , & A new flow cytometric assay for the evaluation of phagocytosis and the oxidative burst in whole blood. J. Immunol. Methods 170, 117–124 (1994).

  5. 5.

    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. Free Radic. Biol. Med. 34, 1359–1368 (2003).

  6. 6.

    et al. Selective fluorescent imaging of superoxide in vivo using ethidium-based probes. Proc. Natl. Acad. Sci. USA 103, 15038–15043 (2006).

  7. 7.

    et al. Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: identification of homo- and heterodimers. Free Radic. Biol. Med. 44, 835–846 (2008).

  8. 8.

    et al. Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence. Proc. Natl. Acad. Sci. USA 102, 5727–5732 (2005).

  9. 9.

    et al. Mitochondrial superoxide production and nuclear factor erythroid 2-related factor 2 activation in p75 neurotrophin receptor-induced motor neuron apoptosis. J. Neurosci. 27, 7777–7785 (2007).

  10. 10.

    et al. Peroxynitrite triggers a phenotypic transformation in spinal cord astrocytes that induces motor neuron apoptosis. J. Neurosci. Res. 67, 21–29 (2002).

  11. 11.

    , & The confounding effects of light, sonication, and Mn(III)TBAP on quantitation of superoxide using hydroethidine. Free Radic. Biol. Med. 41, 1050–1057 (2006).

  12. 12.

    et al. Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology. Biochemistry Mosc. 70, 222–230 (2005).

  13. 13.

    et al. Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry. Nat. Protoc. 2, 2719–2727 (2007).

  14. 14.

    , & Uptake and accumulation of 1-methyl-4-phenylpyridinium by rat liver mitochondria measured using an ion-selective electrode. Biochem. J. 288, 439–443 (1992).

  15. 15.

    , & Mutant SOD1-induced neuronal toxicity is mediated by increased mitochondrial superoxide levels. J. Neurochem. 102, 609–618 (2007).

  16. 16.

    , , , , & Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy. Nat. Protoc. 2, 2295–2301 (2007).

Download references

Author information

Affiliations

  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

Authors

  1. Search for Kristine M Robinson in:

  2. Search for Michael S Janes in:

  3. Search for Joseph S Beckman in:

Competing interests

Mike Janes is an employee of Invitrogen, Inc.

Corresponding author

Correspondence to Joseph S Beckman.

About this article

Publication history

Published

DOI

https://doi.org/10.1038/nprot.2008.56

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.