2.19. Analysis of mitochondrial membrane potential

The mitochondrial membrane potential was analyzed using the mitochondrial membrane potential-sensitive dye tetramethylrhodamine methyl ester (Thermo Fisher; TMRM; 2.5 nM). To normalize to mitochondrial content, hiPSCs were stained with MitoTrackerGreen (Thermo Fisher; 200 nM, 1 h). For that purpose, 5 × 10⁵ hyperoxic or physioxic cultured hiPSCs were plated on gelatine-coated 24 mm glass coverslips (A. Hartenstein, 1001/24) and cultured overnight under hyperoxic (20 % O₂) or physioxic (5 % O₂) conditions. The cells were then cultured in FTDA (supplemented with FGF, TMRM and MitoTrackerGreen) for 1 h at 37 °C, 5 % CO₂ and their respective oxygen concentration. Afterwards, the medium was changed to FTDA that was only supplemented with FGF and TMRM. Measurements were performed on a Zeiss LSM 800 airyscan confocal microscope at basal conditions. TMRM was excited at 561 nm and emission assessed between 580 and 700 nm. MitoTrackerGreen was excited at 488 nm and emission assessed between 500 and 530 nm. The images were processed using ImageJ (version 1.54g). Mean intensity values of TMRM fluorescence (corrected for background) were normalised to mean intensity value of MitoTrackerGreen fluorescence (corrected for background) per image to correct for mitochondrial content. The data derive from n = 5 biological replicates that were normalised to mean values of the basal condition of hyperoxic hiPSCs.