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

Abstract

Depending on its local concentration, hydrogen peroxide (H2O2) can serve as a cellular signaling molecule but can also cause damage to biomolecules. The levels of H2O2 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 H2O2 in cellular signaling, it is crucial to assess its dynamics with high spatiotemporal resolution. Measuring these subcellular H2O2 dynamics has been challenging. However, with the introduction of the super sensitive pH-independent genetically encoded fluorescent H2O2 sensor HyPer7, many limitations of previous measurement approaches could be overcome. Here, we describe a method to measure local H2O2 dynamics in intact human cells, utilizing the HyPer7 sensor in combination with a microscopic multi-mode microplate reader. Graphical abstract: Overview of HyPer7 sensor function and measurement results.