Local Generation and Imaging of Hydrogen Peroxide in Living Cells

Yulia A. Bogdanova1, Carsten Schultz2, Vsevolod V. Belousov3

1 Shemyakin‐Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, 2 Oregon Health & Science University, Portland, 3 Institute for Cardiovascular Physiology, Georg August University Göttingen, Göttingen
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.20
Online Posting Date:  June, 2017
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Described here is a localized H2O2 generation‐detection system consisting of a yeast D‐amino acid oxidase (DAAO) and two spectrally distinct variants of biosensor, HyPer2 and HyPerRed based on circularly permutated yellow and red fluorescent proteins, respectively, which enables spatiotemporal production and examination of the intracellular H2O2 dynamics. The protocol describes using this system in a simple cell culture model. We provide detailed instructions on imaging of H2O2 generated by the activated DAAO. The system can be easily optimized for various combinations of cell types, conditions and DAAO/sensor subcellular localizations. © 2017 by John Wiley & Sons, Inc.

Keywords: biosensors; D‐amino acid oxidase; hydrogen peroxide; HyPer; HyPer2; HyPerRed; metabolic engineering; reactive oxygen species

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: H2O2 Generation in the Nucleus or Cytoplasm and Real‐Time Imaging at Both Compartments
  • Reagents And Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: H2O2 Generation in the Nucleus or Cytoplasm and Real‐Time Imaging at Both Compartments

  • Mammalian cells
  • Appropriate cell culture medium (e.g., complete growth medium; see recipe)
  • Plasmids: HyPer2‐NES (version with nuclear export signal, NES), HyPerRed‐NLS (version with nuclear localization signal, NLS), and one of the DAAO variants: DAAO‐NES or DAAO‐NLS
  • OPTI‐MEM (ThermoFisher Scientific, cat. no. 31985‐047)
  • FuGeneHD transfection reagent (Promega, cat. no. E2311)
  • Tyrode's Salts (Sigma‐Aldrich, cat. no. T2145)
  • HEPES (Promega, cat. no. H5302)
  • Imaging medium (see recipe)
  • H 2O 2 (Sigma–Aldrich, cat. no. 516813)
  • Alanine stock solution (see recipe)
  • Glass‐bottom 35‐mm dishes (MatTek, cat. no. P35G‐1.5‐10‐C)
  • 37°C, 5% CO 2 incubator
  • Wide‐field fluorescent microscope equipped with a thermostatic box
  • Mercury or xenon arc lamp
  • ImageJ or Fiji open software
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Literature Cited

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