Advertisement

Imaging of Intracellular Hydrogen Peroxide Production with HyPer upon Stimulation of HeLa Cells with EGF

  • Kseniya N. Markvicheva
  • Ekaterina A. Bogdanova
  • Dmitry B. Staroverov
  • Sergei Lukyanov
  • Vsevolod V. Belousov
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1990)

Abstract

Reactive oxygen species (ROS) regulate both normal cell functions by activating a number of enzymatic cascades and pathological processes in many diseases by inducing oxidative stress. For many years since the discovery of ROS in biological systems there were no adequate methods of detection and quantification of these molecules inside the living cells. We developed the first genetically encoded fluorescent indicator for intracellular detection of hydrogen peroxide, HyPer, that can be used for imaging of H2O2 production by cells under various physiological and pathological conditions. Unlike most known ROS indicators, HyPer allows for the generation of real-time image series that give precise information about the time course and intensity of H2O2 changes in any compartment of interest. In this chapter we describe the method of confocal imaging of hydrogen peroxide production in HeLa cells upon stimulation with epidermal growth factor. The technique described may be accepted with minimal variations for the use in other cell lines upon various conditions leading to H2O2 production.

Key words

Hydrogen peroxide EGF Fluorescent indicator Fluorescent protein YFP HyPer ROS Confocal microscopy 

Notes

Acknowledgments

This work was supported by grants from the European Commission (FP-6 Integrated Project LSHG-CT-2003-503259), the Russian Academy of Sciences Program in Molecular and Cell Biology, the Russian Foundation for Basic Research (Project 05-04-49316), the National Institutes of Health (GM070358), and the Howard Hughes Medical Institute grant HHMI 55005618.

References

  1. 1.
    Droge W (2002) Free radicals in the physiological control of cell function. Physiol Rev 82:47–95CrossRefGoogle Scholar
  2. 2.
    Crow JP (1997) Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: implications for intracellular measurement of reactive nitrogen and oxygen species. Nitric Oxide 1:145–157CrossRefGoogle Scholar
  3. 3.
    Marchesi E, Rota C, Fann YC, Chignell CF, Mason RP (1999) Photoreduction of the fluorescent dye 2′-7′-dichlorofluorescein: a spin trapping and direct electron spin resonance study with implications for oxidative stress measurements. Free Radic Biol Med 26:148–161CrossRefGoogle Scholar
  4. 4.
    Rota C, Fann YC, Mason RP (1999) Phenoxyl free radical formation during the oxidation of the fluorescent dye 2′,7′-dichlorofluorescein by horseradish peroxidase. Possible consequences for oxidative stress measurements. J Biol Chem 274:28161–28168CrossRefGoogle Scholar
  5. 5.
    Belousov VV, Fradkov AF, Lukyanov KA, Staroverov DB, Shakhbazov KS, Terskikh AV, Lukyanov S (2006) Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 3(4):281–286CrossRefGoogle Scholar
  6. 6.
    Zheng M, Aslund F, Storz G (1998) Activation of the OxyR transcription factor by reversible disulfide bond formation. Science 279:1718–1721CrossRefGoogle Scholar
  7. 7.
    Choi H, Kim S, Mukhopadhyay P, Cho S, Woo J, Storz G, Ryu S (2001) Structural basis of the redox switch in the OxyR transcription factor. Cell 105:103–113CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Kseniya N. Markvicheva
    • 1
  • Ekaterina A. Bogdanova
    • 1
  • Dmitry B. Staroverov
    • 1
  • Sergei Lukyanov
    • 1
    • 2
  • Vsevolod V. Belousov
    • 1
    • 2
  1. 1.Shemyakin-Ovchinnikov Institute of Bioorganic ChemistryMoscowRussian Federation
  2. 2.Pirogov Russian National Research Medical UniversityMoscowRussian Federation

Personalised recommendations