Imaging H2O2 microdomains in receptor tyrosine kinases signaling

Methods Enzymol. 2013;526:175-87. doi: 10.1016/B978-0-12-405883-5.00011-9.

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 H2O2 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Fluorescent Dyes / analysis*
  • Fluorescent Dyes / metabolism
  • Humans
  • Hydrogen Peroxide / analysis*
  • Hydrogen Peroxide / metabolism
  • Image Processing, Computer-Assisted / methods
  • Microscopy, Confocal / methods
  • Microscopy, Fluorescence / methods
  • Optical Imaging / methods*
  • Receptor Protein-Tyrosine Kinases / metabolism*
  • Signal Transduction*
  • Transfection / methods

Substances

  • Fluorescent Dyes
  • Hydrogen Peroxide
  • Receptor Protein-Tyrosine Kinases