NADPH Oxidases pp 259-274 | Cite as

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

  • Yulia G. Ermakova
  • Nataliya M. Mishina
  • Carsten Schultz
  • Vsevolod V. BelousovEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1982)


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.

Key words

ROS Hydrogen peroxide HyPer HyPerRed PDGF D-amino acid oxidase DAAO Ratiometric imaging Microscopy 



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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Yulia G. Ermakova
    • 1
    • 2
  • Nataliya M. Mishina
    • 1
  • Carsten Schultz
    • 2
    • 3
  • Vsevolod V. Belousov
    • 1
    • 4
    • 5
    Email author
  1. 1.Shemyakin-Ovchinnikov Institute of Bioorganic ChemistryMoscowRussia
  2. 2.European Molecular Biology LaboratoryHeidelbergGermany
  3. 3.Oregon Health and Science UniversityPortlandUSA
  4. 4.Pirogov Russian National Research Medical UniversityMoscowRussia
  5. 5.Georg August University of GöttingenGöttingenGermany

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