Hydrogen Peroxide Imaging via HyPer7 Probe in Adeno Associated Virus-Transduced Primary Mouse Hepatocytes

Hydrogen peroxide (H₂O₂) plays an important role in liver metabolism as a second messenger under physiological conditions, as well as in the development of oxidative stress and pathology. The major metabolic functions of the liver are carried out by hepatocytes, the parenchymal cells that constitute most of the liver mass. The study of oxidative stress in the liver, in particular the determination of a causal relationship between pathological progression and H₂O₂ production, requires an optimal model system. The chemogenetic system based on yeast d-amino acid oxidase (DAAO) fused to the C-terminus of the HyPer7 biosensor has been established as a tool for controlled H₂O₂ production and examination of intracellular H₂O₂ dynamics in vitro and in vivo. This article details adeno associated virus (AAV)-mediated targeted delivery of the HyPer7-DAAO transgene into mouse liver, followed by isolation of primary hepatocytes for quantitative evaluation of intracellular H₂O₂ dynamics based on ratiometric HyPer7 fluorescence acquired by wide-field microscopy and subsequent image analysis. © 2026 Wiley Periodicals LLC. Basic Protocol 1: AAVs production and purification Basic Protocol 2: Intraparenchymal injection of AAVs into mouse liver Basic Protocol 3: Primary mouse hepatocyte culture preparation Basic Protocol 4: Wide-field fluorescence microscopy of HyPer7 in primary mouse hepatocyte culture Basic Protocol 5: Time series processing.