Measuring Intracellular H 2 O 2 in Intact Human Cells Using the Genetically Encoded Fluorescent Sensor HyPer7

Lianne J H C Jacobs; Michaela N Hoehne; Jan Riemer

doi:10.21769/BioProtoc.4538

Measuring Intracellular H ₂ O ₂ in Intact Human Cells Using the Genetically Encoded Fluorescent Sensor HyPer7

Bio Protoc. 2022 Oct 20;12(20):e4538. doi: 10.21769/BioProtoc.4538.

Authors

Lianne J H C Jacobs¹, Michaela N Hoehne¹, Jan Riemer^{1

2}

Affiliations

¹ Institute of Biochemistry, Redox Biochemistry, University of Cologne, Zuelpicher Str. 47a/R. 3.49, 50674 Cologne, Germany.
² Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50931 Cologne, Germany.

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 H ₂O₂ 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 H₂O₂sensor 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.

Keywords: H2O2 measurement; HyPer7; Real-time imaging; Subcellular resolution; pH-independent.