Cytoplasmic Hydrogen Peroxide Imaging

The plasmid for the mammalian expression of cytoplasm targeted ultrasensitive hydrogen peroxide indicator HyPer7 for optical imaging (pCS2+HyPer7-NES) was a generous gift from Vsevolod Belousov (IBCh, Moscow, Russia) (Addgene plasmid # 136467; http://n2t.net/addgene:136467 (accessed on 9 June 2022; RRID: Addgene_136467) [40 (link)]. Mesothelial and MPM cells were seeded into 2 mL plastic dishes, and HyPer7-NES transfection (3 μg DNA / dish) was performed when cells reached the 60–70% confluency using the JetOPTIMUS DNA transfection reagent (# 117-15, Polyplus transfection, Illkirch-Graffenstaden, France) according to the manufacturer’s instructions. Briefly, the medium was removed and substituted with Opti-MEM containing the plasmid DNA and the transfection reagent. Plasmid DNA (3 μg) was diluted in JetOPTIMUS Buffer (# 717-60, Polyplus transfection, Illkirch-Graffenstaden, France) and then mixed with JetOPTIMUS reagent according to the suggested ratio 1:1 between μg of DNA and μL of transfection reagent. After 10 min incubation at room temperature, the solution containing the DNA was added drop-wise to the cells and incubated at 37 °C for 4 h. Then, the media were exchanged for fresh culture media. All the experiments were performed 24 h after transfection.