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Visualization of Intracellular Hydrogen Peroxide with the Genetically Encoded Fluorescent Probe HyPer in NIH-3T3 Cells

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NADPH Oxidases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1982))

Abstract

Reactive oxygen species (ROS) are involved in regulating normal physiological cell functions as second messengers as well as nonspecific damage of biomolecules in a pathological process known as oxidative stress. The HyPer family of genetically encoded probes are a useful noninvasive tool for monitoring the real-time dynamics of ROS in individual cells or model organisms. HyPer, the first genetically encoded probe for detection of hydrogen peroxide (H2O2), is oxidized with high specificity and sensitivity by H2O2, leading to ratiometric changes in the fluorescence excitation spectrum of the probe. These changes can be detected with a wide range of commercial confocal and wide-field microscope systems. Here we describe a detailed protocol for ratiometric monitoring of H2O2 produced by D-amino acid oxidase (DAAO) or by NADPH oxidase (NOX) in NIH-3T3 cells using the HyPer probe.

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Acknowledgment

This work was supported by the Russian Science Foundation grant 17-14-01086 (experiments with D-amino acid oxidase), Russian Foundation for Basic Research grant 18-54-74003 (image processing and PDGF experiments), and DFG IRTG 1816. YGE received Sergey Shpiz fellowship. Experiments were partially carried out using the equipment provided by the IBCH сore facility (CKP IBCH, supported by the Russian Ministry of Education and Science, grant RFMEFI62117X0018).

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Authors and Affiliations

  1. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia

    Yulia G. Ermakova, Nataliya M. Mishina & Vsevolod V. Belousov

  2. European Molecular Biology Laboratory, Heidelberg, Germany

    Yulia G. Ermakova & Carsten Schultz

  3. Oregon Health and Science University, Portland, OR, USA

    Carsten Schultz

  4. Pirogov Russian National Research Medical University, Moscow, Russia

    Vsevolod V. Belousov

  5. Georg August University of Göttingen, Göttingen, Germany

    Vsevolod V. Belousov

Authors
  1. Yulia G. Ermakova
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  2. Nataliya M. Mishina
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  3. Carsten Schultz
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  4. Vsevolod V. Belousov
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Corresponding author

Correspondence to Vsevolod V. Belousov .

Editor information

Editors and Affiliations

  1. Conway Institute, School of Medicine, University College Dublin, Dublin, Ireland

    Ulla G. Knaus

  2. Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Thomas L. Leto

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Ermakova, Y.G., Mishina, N.M., Schultz, C., Belousov, V.V. (2019). Visualization of Intracellular Hydrogen Peroxide with the Genetically Encoded Fluorescent Probe HyPer in NIH-3T3 Cells. In: Knaus, U., Leto, T. (eds) NADPH Oxidases. Methods in Molecular Biology, vol 1982. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9424-3_15

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  • DOI: https://doi.org/10.1007/978-1-4939-9424-3_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9423-6

  • Online ISBN: 978-1-4939-9424-3

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