Measuring Intracellular H2O2 in Intact Human Cells Using the Genetically Encoded Fluorescent Sensor HyPer7

Depending on its local concentration, hydrogen peroxide (HO) can serve as a cellular signaling molecule but can also cause damage to biomolecules. The levels of H O are influenced by the activity of its generator sites, local antioxidative systems, and the metabolic state of the cell. To study and understand the role of HO in cellular signaling, it is crucial to assess its dynamics with high spatiotemporal resolution. Measuring these subcellular HO dynamics has been challenging. However, with the introduction of the super sensitive pH-independent genetically encoded fluorescent HOsensor HyPer7, many limitations of previous measurement approaches could be overcome. Here, we describe a method to measure local HO dynamics in intact human cells, utilizing the HyPer7 sensor in combination with a microscopic multi-mode microplate reader. Graphical abstract: