The selective detection of mitochondrial superoxide by live cell imaging (original) (raw)

Nature Protocols volume 3, pages 941–947 (2008)Cite this article

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.

This is a preview of subscription content, access via your institution

Access options

Subscribe to this journal

Receive 12 print issues and online access

$259.00 per year

only $21.58 per issue

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Additional access options:

Similar content being viewed by others

References

  1. Shigenaga, M.K., Hagen, T.M. & Ames, B.N. Oxidative damage and mitochondrial decay in aging. Proc. Natl. Acad. Sci. USA 91, 10771–10778 (1994).
    Article CAS Google Scholar
  2. Green, K., Brand, M.D. & Murphy, M.P. Prevention of mitochondrial oxidative damage as a therapeutic strategy in diabetes. Diabetes 53 (suppl 1): S110–118 (2004).
    Article CAS Google Scholar
  3. Rothe, G. & Valet, G. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2′,7′-dichlorofluorescin. J. Leukoc. Biol. 47, 440–448 (1990).
    Article CAS Google Scholar
  4. Perticarari, S., Presani, G. & Banfi, E. A new flow cytometric assay for the evaluation of phagocytosis and the oxidative burst in whole blood. J. Immunol. Methods 170, 117–124 (1994).
    Article CAS Google Scholar
  5. Zhao, H. 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).
    Article CAS Google Scholar
  6. Robinson, K.M. et al. Selective fluorescent imaging of superoxide in vivo using ethidium-based probes. Proc. Natl. Acad. Sci. USA 103, 15038–15043 (2006).
    Article CAS Google Scholar
  7. Zielonka, J. 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).
    Article CAS Google Scholar
  8. Zhao, H. 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).
    Article CAS Google Scholar
  9. Pehar, M. 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).
    Article CAS Google Scholar
  10. Cassina, P. et al. Peroxynitrite triggers a phenotypic transformation in spinal cord astrocytes that induces motor neuron apoptosis. J. Neurosci. Res. 67, 21–29 (2002).
    Article CAS Google Scholar
  11. Zielonka, J., Vasquez-Vivar, J. & Kalyanaraman, B. The confounding effects of light, sonication, and Mn(III)TBAP on quantitation of superoxide using hydroethidine. Free Radic. Biol. Med. 41, 1050–1057 (2006).
    Article CAS Google Scholar
  12. Ross, M.F. et al. Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology. Biochemistry Mosc. 70, 222–230 (2005).
    Article CAS Google Scholar
  13. Troiano, L. et al. Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry. Nat. Protoc. 2, 2719–2727 (2007).
    Article CAS Google Scholar
  14. Davey, G.P., Tipton, K.F. & Murphy, M.P. Uptake and accumulation of 1-methyl-4-phenylpyridinium by rat liver mitochondria measured using an ion-selective electrode. Biochem. J. 288, 439–443 (1992).
    Article CAS Google Scholar
  15. Zimmerman, M.C., Oberley, L.W. & Flanagan, S.W. Mutant SOD1-induced neuronal toxicity is mediated by increased mitochondrial superoxide levels. J. Neurochem. 102, 609–618 (2007).
    Article CAS Google Scholar
  16. Mukhopadhyay, P., Rajesh, M.,, Haskó, G., Hawkins, B.J., Madesh, M., & Pacher, P. Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy. Nat. Protoc. 2, 2295–2301 (2007).
    Article CAS Google Scholar

Download references

Author information

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

Authors

  1. Kristine M Robinson
    You can also search for this author inPubMed Google Scholar
  2. Michael S Janes
    You can also search for this author inPubMed Google Scholar
  3. Joseph S Beckman
    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence toJoseph S Beckman.

Ethics declarations

Competing interests

Mike Janes is an employee of Invitrogen, Inc.

Rights and permissions

About this article

Cite this article

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

Download citation

This article is cited by