NADPH Oxidases pp 275-282 | Cite as

Imaging Intracellular H2O2 with the Genetically Encoded PerFRET and OxyFRET Probes

  • Balázs EnyediEmail author
  • Miklós GeisztEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1982)


Hydrogen peroxide (H2O2) is an important signaling intermediate with various regulatory and effector functions. Despite its significance, the subcellular organization of H2O2 signals is poorly understood. Introducing novel techniques for the intracellular detection of H2O2 would be essential for a more complete understanding of its role in cellular signaling. We previously reported the development of two novel fluorescence resonance energy transfer (FRET)-based protein sensors that showed opposite emission ratio changes upon reaction with H2O2. In this chapter, we detail the methods for using OxyFRET and PerFRET for the assessment of changes in subcellular H2O2 levels.

Key words

Hydrogen peroxide PerFRET OxyFRET Genetically encoded sensor Fluorescent biosensor imaging 



This work was supported by grants from the Hungarian Research Fund (OTKA NF72669) and from the National Office for Research and Technology (OMFB-01680/2009). The work was also financed by the Higher Education Institutional Excellence Programme (FIKP) of the Ministry of Human Capacities in Hungary, within the framework of the Molecular Biology thematic programme of the Semmelweis University and by “Lendület” grants from the Hungarian Academy of Sciences to both MG (LP2011-001/2011) and BE (LP2018-13/2018). EB is also grateful for the support of the Premium postdoctoral research program (PPD-016/2017) of the Hungarian Academy of Sciences.


  1. 1.
    Murphy MP, Holmgren A, Larsson NG et al (2011) Unraveling the biological roles of reactive oxygen species. Cell Metab 13:361–366CrossRefGoogle Scholar
  2. 2.
    Lambeth JD, Neish AS (2014) Nox enzymes and new thinking on reactive oxygen: a double-edged sword revisited. Annu Rev Pathol 9:119–145CrossRefGoogle Scholar
  3. 3.
    Sirokmány G, Donkó Á, Geiszt M (2016) Nox/duox family of NADPH oxidases: lessons from knockout mouse models. Trends Pharmacol Sci 37:318–327CrossRefGoogle Scholar
  4. 4.
    Kalyanaraman B, Darley-Usmar V, Davies KJ et al (2012) Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations. Free Radic Biol Med 52:1–6CrossRefGoogle Scholar
  5. 5.
    Bilan DS, Belousov VV (2017) New tools for redox biology: from imaging to manipulation. Free Radic Biol Med 109:167–188CrossRefGoogle Scholar
  6. 6.
    Enyedi B, Várnai P, Geiszt M (2010) Redox state of the endoplasmic reticulum is controlled by ero1l-alpha and intraluminal calcium. Antioxid Redox Signal 13:721–729CrossRefGoogle Scholar
  7. 7.
    Gutscher M, Sobotta MC, Wabnitz GH et al (2009) Proximity-based protein thiol oxidation by H2O2-scavenging peroxidases. J Biol Chem 284:31532–31540CrossRefGoogle Scholar
  8. 8.
    Belousov VV, Fradkov AF, Lukyanov KA et al (2006) Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 3:281–286CrossRefGoogle Scholar
  9. 9.
    Albrecht SC, Barata AG, Grosshans J et al (2011) In vivo mapping of hydrogen peroxide and oxidized glutathione reveals chemical and regional specificity of redox homeostasis. Cell Metab 14:819–829CrossRefGoogle Scholar
  10. 10.
    Enyedi B, Zana M, Donkó A, Geiszt M (2013) Spatial and temporal analysis of NADPH oxidase-generated hydrogen peroxide signals by novel fluorescent reporter proteins. Antioxid Redox Signal 19:523–534CrossRefGoogle Scholar
  11. 11.
    Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. CrossRefGoogle Scholar
  12. 12.
    Whitaker M (2010) Genetically encoded probes for measurement of intracellular calcium. Methods Cell Biol 99:153–182. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Faculty of Medicine, Department of PhysiologySemmelweis UniversityBudapestHungary
  2. 2.MTA-SE Lendﺲlet Tissue Damage Research GroupHungarian Academy of Sciences and Semmelweis UniversityBudapestHungary

Personalised recommendations