Imaging of intracellular hydrogen peroxide production with HyPer upon stimulation of HeLa cells with epidermal growth factor.
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 the 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 the generation of a 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.
Markvicheva
Kseniya N
KN
Laboratory of Molecular Technologies for Biology and Medicine, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
Bogdanova
Ekaterina A
EA
Staroverov
Dmitry B
DB
Lukyanov
Sergei
S
Belousov
Vsevolod V
VV
D004815:Epidermal Growth Factor / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D042541:Intracellular Space / Q000187:drug effects* / Q000378:metabolism*; D014162:Transfection
D004815:Epidermal Growth Factor; D006861:Hydrogen Peroxide