Live-Cell Assessment of Mitochondrial Reactive Oxygen Species Using Dihydroethidine

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


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 



Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone








Reactive oxygen species




Mitochondrial membrane potential



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.


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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

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