Imaging H2O2 microdomains in receptor tyrosine kinases signaling

Nataliya M. Mishina, Kseniya N. Markvicheva, Arkady F. Fradkov, Elena V. Zagaynova, Carsten Schultz, Sergey Lukyanov, Vsevolod V. Belousov

Research output: Chapter in Book/Report/Conference proceedingChapter

11 Scopus citations

Abstract

HyPer, a ratiometric genetically encoded fluorescent sensor, is a popular tool for intracellular hydrogen peroxide detection. When expressed in cultured cells, the freely diffusing version of the sensor (HyPer-cyto) detects temporal patterns of H2O2 generation. However, rapid diffusion of the probe within the nucleocytoplasmic compartment averages the H 2O2 signal even in cases of local oxidant production. Consequently, we immobilized the sensor within specific subcellular compartments allowing it to monitor local increases in H2O2. Here, we provide a protocol of ratiometric imaging and ImageJ-based quantification of H2O2 microdomains produced by cells upon physiological stimulation.

Original languageEnglish (US)
Title of host publicationHydrogen Peroxide and Cell Signaling, Part A
PublisherAcademic Press Inc.
Pages175-187
Number of pages13
ISBN (Print)9780124058835
DOIs
StatePublished - 2013

Publication series

NameMethods in Enzymology
Volume526
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • HyPer
  • Hydrogen peroxide microdomains
  • Imaging
  • NADPH oxidases
  • Receptor tyrosine kinases
  • Sensor
  • Signaling

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Mishina, N. M., Markvicheva, K. N., Fradkov, A. F., Zagaynova, E. V., Schultz, C., Lukyanov, S., & Belousov, V. V. (2013). Imaging H2O2 microdomains in receptor tyrosine kinases signaling. In Hydrogen Peroxide and Cell Signaling, Part A (pp. 175-187). (Methods in Enzymology; Vol. 526). Academic Press Inc.. https://doi.org/10.1016/B978-0-12-405883-5.00011-9