Local Generation and Imaging of Hydrogen Peroxide in Living Cells

Described here is a localized H₂ O₂ 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 H₂ O₂ dynamics. The protocol describes using this system in a simple cell culture model. We provide detailed instructions on imaging of H₂ O₂ 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.