Measuring intracellular concentration of hydrogen peroxide with the use of genetically encoded H(2)O(2) 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 H(2)O(2) biosensor HyPer. The method utilizes flow cytometric measurements of HyPer fluorescence in H(2)O(2)-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 H(2)O(2). Applying this method to K562 cells, we have estimated the gradient as about 390-fold between the extracellular and intracellular level of exogenous H(2)O(2) in cells exposed to the micromole doses of peroxide, as well as the average basal level of H(2)O(2) in the cytosol of undisturbed cells ([Formula: see text]). The method can be extended to other H(2)O(2)-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 H(2)O(2) concentrations but also the concentration of peroxide build up in the vicinity of redox probes.