Imaging of Intracellular Hydrogen Peroxide Production with HyPer upon Stimulation of HeLa Cells with EGF
Reactive oxygen species (ROS) regulate both normal cell functions by activating a number of enzymatic cascades and pathological processes in many diseases by inducing oxidative stress. For many years since the discovery of ROS in biological systems there were no adequate methods of detection and quantification of these molecules inside the living cells. We developed the first genetically encoded fluorescent indicator for intracellular detection of hydrogen peroxide, HyPer, that can be used for imaging of H2O2 production by cells under various physiological and pathological conditions. Unlike most known ROS indicators, HyPer allows for the generation of real-time image series that give precise information about the time course and intensity of H2O2 changes in any compartment of interest. In this chapter we describe the method of confocal imaging of hydrogen peroxide production in HeLa cells upon stimulation with epidermal growth factor. The technique described may be accepted with minimal variations for the use in other cell lines upon various conditions leading to H2O2 production.
Key wordsHydrogen peroxide EGF Fluorescent indicator Fluorescent protein YFP HyPer ROS Confocal microscopy
This work was supported by grants from the European Commission (FP-6 Integrated Project LSHG-CT-2003-503259), the Russian Academy of Sciences Program in Molecular and Cell Biology, the Russian Foundation for Basic Research (Project 05-04-49316), the National Institutes of Health (GM070358), and the Howard Hughes Medical Institute grant HHMI 55005618.