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Quantitative Monitoring of Subcellular Redox Dynamics in Living Mammalian Cells Using RoGFP2-Based Probes

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Peroxisomes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1595))

Abstract

To gain additional insight into how specific cell organelles may participate in redox signaling, it is essential to have access to tools and methodologies that are suitable to monitor spatiotemporal differences in the levels of different reactive oxygen species (ROS) and the oxidation state of specific redox couples. Over the years, the use of genetically encoded fluorescent redox indicators with a ratiometric readout has constantly gained in popularity because they can easily be targeted to various subcellular compartments and monitored in real time in single cells. Here we provide step-by-step protocols and tips for the successful use of roGFP2, a redox-sensitive variant of the enhanced green fluorescent protein, to monitor changes in glutathione redox balance and hydrogen peroxide homeostasis in the cytosol, peroxisomes, and mitochondria of mammalian cells.

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Acknowledgments

This work was supported by grants from the KU Leuven (OT/14/100) and the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (Onderzoeksproject G095315 N).

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Authors and Affiliations

  1. Laboratory of Lipid Biochemistry and Protein Interactions, Department of Cellular and Molecular Medicine, University of Leuven - KU Leuven, Herestraat 49 - box 601, Leuven, 3000, Belgium

    Celien Lismont, Paul A. Walton & Marc Fransen

  2. Department of Anatomy and Cell Biology, University of Western Ontario, London, Canada

    Paul A. Walton

Authors
  1. Celien Lismont
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  2. Paul A. Walton
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  3. Marc Fransen
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Corresponding author

Correspondence to Marc Fransen .

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Editors and Affiliations

  1. College of Life and Environmental Sciences, Biosciences, University of Exeter, Exeter, United Kingdom

    Michael Schrader

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Lismont, C., Walton, P.A., Fransen, M. (2017). Quantitative Monitoring of Subcellular Redox Dynamics in Living Mammalian Cells Using RoGFP2-Based Probes. In: Schrader, M. (eds) Peroxisomes. Methods in Molecular Biology, vol 1595. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6937-1_14

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  • DOI: https://doi.org/10.1007/978-1-4939-6937-1_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6935-7

  • Online ISBN: 978-1-4939-6937-1

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