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. Belousov

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages259-274
Number of pages16
DOIs
StatePublished - Jan 1 2019

Publication series

NameMethods in Molecular Biology
Volume1982
ISSN (Print)1064-3745

Fingerprint

NIH 3T3 Cells
Fluorescent Dyes
Hydrogen Peroxide
Reactive Oxygen Species
D-Amino-Acid Oxidase
NADPH Oxidase
Second Messenger Systems
Pathologic Processes
Oxidative Stress
Fluorescence
Sensitivity and Specificity

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

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 Methods in Molecular Biology (pp. 259-274). (Methods in Molecular Biology; Vol. 1982). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-9424-3_15

Visualization of intracellular hydrogen peroxide with the genetically encoded fluorescent probe HyPer in NIH-3T3 cells. / Ermakova, Yulia G.; Mishina, Nataliya M.; Schultz, Carsten; Belousov, Vsevolod V.

Methods in Molecular Biology. Humana Press Inc., 2019. p. 259-274 (Methods in Molecular Biology; Vol. 1982).

Research output: Chapter in Book/Report/Conference proceedingChapter

Ermakova, YG, Mishina, NM, Schultz, C & Belousov, VV 2019, Visualization of intracellular hydrogen peroxide with the genetically encoded fluorescent probe HyPer in NIH-3T3 cells. in Methods in Molecular Biology. Methods in Molecular Biology, vol. 1982, Humana Press Inc., pp. 259-274. https://doi.org/10.1007/978-1-4939-9424-3_15
Ermakova YG, Mishina NM, Schultz C, Belousov VV. Visualization of intracellular hydrogen peroxide with the genetically encoded fluorescent probe HyPer in NIH-3T3 cells. In Methods in Molecular Biology. Humana Press Inc. 2019. p. 259-274. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-9424-3_15
Ermakova, Yulia G. ; Mishina, Nataliya M. ; Schultz, Carsten ; Belousov, Vsevolod V. / Visualization of intracellular hydrogen peroxide with the genetically encoded fluorescent probe HyPer in NIH-3T3 cells. Methods in Molecular Biology. Humana Press Inc., 2019. pp. 259-274 (Methods in Molecular Biology).
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