Advertisement

Live-Cell Assessment of Mitochondrial Reactive Oxygen Species Using Dihydroethidine

  • Marleen Forkink
  • Peter H. G. M. Willems
  • Werner J. H. Koopman
  • Sander Grefte
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1264)

Abstract

Reactive oxygen species (ROS) play an important role in both physiology and pathology. Mitochondria are an important source of the primary ROS superoxide. However, accurate detection of mitochondrial superoxide especially in living cells remains a difficult task. Here, we describe a method and the pitfalls to detect superoxide in both mitochondria and the entire cell using dihydroethidium (HEt) and live-cell microscopy.

Key words

MitoSOX Membrane potential Imaging 

Abbreviations

FCCP

Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone

HEt

Dihydroethidium

HT

HEPES-Tris

mito-HEt

Mito-dihydroethidium

ROS

Reactive oxygen species

TPP

Triphenylphosphonium

Δψ

Mitochondrial membrane potential

Notes

Acknowledgments

This research was supported by a grant from the Netherlands Organization for Scientific Research (NWO, No: 911-02-008), the Energy4All Foundation, the NWO Centers for Systems Biology Research initiative (CSBR09/013V), and a grant from the Institute for Genetic and Metabolic Disease (IGMD) of the Radboud University Medical Center (RUMC) to W.J.H.K. We are grateful to Dr.A. S. De Jong (Dept. of Biochemistry, RUMC) for performing the HEt and mito-HEt experiments on human skin fibroblasts.

References

  1. 1.
    Finkel T (2012) Signal transduction by mitochondrial oxidants. J Biol Chem 287:4434–4440PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Murphy MP, Holmgren A, Larsson NG (2011) Unraveling the biological roles of reactive oxygen species. Cell Metab 13:361–366PubMedCrossRefGoogle Scholar
  3. 3.
    Distelmaier F, Valsecchi F, Forkink M et al (2012) Trolox-sensitive reactive oxygen species regulate mitochondrial morphology, oxidative phosphorylation and cytosolic calcium handling in healthy cells. Antioxid Redox Signal 17:1657–1669PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Brown GC, Borutaite V (2012) There is no evidence that mitochondria are the main source of reactive oxygen species in mammalian cells. Mitochondrion 12:1–4PubMedCrossRefGoogle Scholar
  5. 5.
    Zhou L, Aon M, Almas T et al (2010) A reaction–diffusion model of ROS-induced ROS release in a mitochondrial network. PLoS Comput Biol 6:e1000657PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Tormos KV, Anso E, Hamanaka RB et al (2011) Mitochondrial complex III ROS regulate adipocyte differentiation. Cell Metab 14:537–544PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Murphy M (2009) How mitochondria produce reactive oxygen species. Biochem J 417:1–13PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Wang W, Fang H, Groom L et al (2008) Superoxide flashes in single mitochondria. Cell 134:279–290PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Pouvreau S (2010) Superoxide flashes in mouse skeletal muscle are produced by discrete arrays of active mitochondria operating coherently. PLoS One 5:e13035PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Fang H, Chen M, Ding Y et al (2011) Imaging superoxide flash and metabolism-coupled mitochondrial permeability transition in living animals. Cell Res 21:1295–1304PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Muller FL (2009) A critical evaluation of cpYFP as a probe for superoxide. Free Radic Biol Med 47:1779–1780PubMedCrossRefGoogle Scholar
  12. 12.
    Schwarzländer M, Murphy MP, Duchen MR et al (2012) Mitochondrial “flashes”: a radical concept repHined. Trends Cell Biol 22:503–508PubMedCrossRefGoogle Scholar
  13. 13.
    Wei-Lapierre L, Gong G, Gerstner BJ et al (2013) Respective contribution of mitochondrial superoxide and pH to Mt-cpYFP flash activity. J Biol Chem 288:10567–10577PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Zhao H, Kalivendi S, Zhang H et al (2003) 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 34:1359–1368PubMedCrossRefGoogle Scholar
  15. 15.
    Zhao H, Joseph J, Fales HM et al (2005) Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence. Proc Natl Acad Sci U S A 102:5727–5732PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Robinson KM, Janes MS, Pehar M et al (2006) Selective fluorescent imaging of superoxide in vivo using ethidium-based probes. Proc Natl Acad Sci U S A 103:15038–15043PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Benov L, Sztejnberg L, Fridovich I (1998) Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical. Free Radic Biol Med 25:826–831PubMedCrossRefGoogle Scholar
  18. 18.
    Ince C, Beekman RE, Verschragen G (1990) A micro-perfusion chamber for single-cell fluorescence measurements. J Immunol Methods 128:227–234PubMedCrossRefGoogle Scholar
  19. 19.
    Forkink M, Smeitink JAM, Brock R et al (2010) Detection and manipulation of mitochondrial reactive oxygen species in mammalian cells. Biochim Biophys Acta 1797:1034–1044PubMedCrossRefGoogle Scholar
  20. 20.
    Koopman W, Verkaart S, Visch H et al (2005) Inhibition of complex I of the electron transport chain causes O2-mediated mitochondrial outgrowth. Am J Physiol Cell Physiol 288:C1440–C1450PubMedCrossRefGoogle Scholar
  21. 21.
    Zielonka J, Vasquez-Vivar J, Kalyanaraman B (2008) Detection of 2-hydroxyethidium in cellular systems: a unique marker product of superoxide and hydroethidine. Nat Protoc 3:8–21PubMedCrossRefGoogle Scholar
  22. 22.
    Zielonka J, Kalyanaraman B (2010) Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: another inconvenient truth. Free Radic Biol Med 48:983–1001PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Marleen Forkink
    • 1
  • Peter H. G. M. Willems
    • 1
  • Werner J. H. Koopman
    • 1
  • Sander Grefte
    • 1
  1. 1.Department of Biochemistry, Raboud Institute for Molecular Life SciencesRadboud University Medical CentreNijmegenThe Netherlands

Personalised recommendations