Measuring intracellular concentration of hydrogen peroxide with the use of genetically encoded H₂O₂ biosensor HyPer

In this study, we propose a method for quantification of average hydrogen peroxide concentration within a living cell that is based on the use of genetically encoded H₂O₂ biosensor HyPer. The method utilizes flow cytometric measurements of HyPer fluorescence in H₂O₂-exposed cells to analyze the biosensor oxidation kinetics. Fitting the experimental curves with kinetic equations allows determining the rate constants of HyPer oxidation/reduction which are used further for the calculation of peroxide concentrations in the cells of interest both in the presence and absence of external H₂O₂. Applying this method to K562 cells, we have estimated the gradient as about 390-fold between the extracellular and intracellular level of exogenous H₂O₂ in cells exposed to the micromole doses of peroxide, as well as the average basal level of H₂O₂ in the cytosol of undisturbed cells ( [Formula: see text] ). The method can be extended to other H₂O₂-sensitive redox probes or to procedures in which, rather than adding external peroxide, intracellular production of peroxide is triggered, providing a tool to quantitate not only basal average H₂O₂ concentrations but also the concentration of peroxide build up in the vicinity of redox probes.