130 Chapter 6 antibodies (REA, Miltenyi Biotec, 130-096-932) were used. Media and washes were collected to obtain a complete representation, including detached cells. The samples were analysed using a FACS Canto system (BD Bioscience, FACSDiva software 6.0) and FlowJo software (BD Bioscience, v10). Beating rate analysis. iPSC-CM beating rate was microscopically determined (Olympus CKX53) and recorded (Hero 8 GoPro camera). Beats per minute were quantified by counting the number of contractions of individual cells from 10 second videos. Seahorse analysis. Mitochondrial respiration was measured using a XF24 Extracellular Flux Analyzer (Seahorse Bioscience, Agilent) to assess the effect of hypoxia on the electron transport chain complexes. XF24 plates (Agilent, 103518-100) were used and coated with 0.1 mg/mL Matrigel (Corning, CLS356252) before cells were seeded at a density of 1,0x105 cells per well. 24 hours or 4 hours before Seahorse analysis, plates were placed in a GasPak EZ Pouch system to induce hypoxia following manufacturer’s instructions. One hour before measurement cells were washed three times with Seahorse XF DMEM Basal Medium (Agilent, 103680-100), supplemented with 2% B27, 4 mM glutamine (Gibco, 25030081), 10 mM glucose (ThermoFisher Scientific, 15023021), and 1% chemically defined lipid concentrate (ThermoFisher Scientific, 11905031). Oxygen consumption rate (OCR) was determined using XF Cell Mito Stress Assay (Agilent, 103015-100) with subsequent additions of: (1) ATP synthase inhibitor: 2.5 mM oligomycin, (2) uncoupler: 2.5 mM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) and (3) Complex I/II inhibitors: 2.5 mM Rotenone/Antimycin A. At the end of measurement, the oxygen consumption rate (OCR) values were normalized to cell numbers per well as assessed by Hoechst 33342 (Thermofisher Scientific) staining at 20x magnification imaging using the Evos microscope and ImageJ29. Baseline respiration was calculated by subtracting the OCR, after addition of rotenone and antimycin A, from the respiration as measured at the first time point. ATP production was calculated as the OCR at the first time point minus the OCR after oligomycin infusion. The proton leak was determined by subtracting the OCR after FCCP infusion from the value after oligomycin infusion. Maximal OCR was calculated as the difference between the OCR after FCCP infusion and after rotenone and antimycin A infusion. Respiratory capacity was calculated by subtracting the difference between the OCR before the addition of inhibitors and after rotenone and antimycin A infusion from the OCR after FCCP infusion. Lastly, non-mitochondrial respiration was defined as the OCR after rotenone and antimycin A infusion. The experiment was done in 1-2 biological replicates, with each replicate consisting of 5 technical repeats per condition. Statistical analysis. iPSC lines from two donors were used and the number (N) depicted in individual figures represents the number of independent experiments performed. Each
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