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.
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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
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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.
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Forkink, M., Willems, P.H.G.M., Koopman, W.J.H., Grefte, S. (2015). Live-Cell Assessment of Mitochondrial Reactive Oxygen Species Using Dihydroethidine. In: Weissig, V., Edeas, M. (eds) Mitochondrial Medicine. Methods in Molecular Biology, vol 1264. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2257-4_15
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DOI: https://doi.org/10.1007/978-1-4939-2257-4_15
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