129 Metabolic maturation increases susceptibility to hypoxia-induced damage in human iPSC-derived cardiomyocytes 6 Preconditioning protective treatment. Before hypoxia, the medium was changed to media of variant nutrient composition (Supplementary table 1). iPSC-CMs were preconditioned with 60 µM necroptosis inhibitor Necrostatin-1 (Nec-1, Abcam ab141053) for 24 hours. Lactate dehydrogenase, glucose and lactate measurements. Media glucose and lactate levels were determined using the Accutrend plus device (Roche, 05050472023) and verified using the glucose (Sigma, MAK083-1KT) and lactate colorimetric kits (Sigma, MAK064-1KT). Lactate dehydrogenase levels were determined using the LDH assay colorimetric kit (Abcam, ab102526) according to manufacturer’s instructions. Immunofluorescent staining. Live dead assay was performed using EthD1/calcein AM staining with the LIVE/DEAD Kit, for mammalian cells (ThermoFisher Scientific, L3224) according to manufacturer’s instructions and imaging was performed using the EVOS Floid microscope (ThermoFisher Scientific, AMF5000). The ratio of EthD1 to Calcein AM was determined by dividing the number of EthD1-positive cells by the number of Calcein AM-positive cells per image. The averages of 5 images were used per condition. For immunofluorescence stainings, after fixation in 4% paraformaldehyde for 30 minutes, cells were permeabilized in 0,1% Triton-X100 (Sigma, 11332481001) before blocking in 10% normal goat serum/1% BSA (Sigma, A9418). Primary antibodies used were: ACTN1 (Sigma-Aldrich, A7811, 1:200), cardiac troponin T (Abcam, ab45932, 1:100), Ki-67 (Abcam, ab8330, 1:200), pH3 (Cell Signaling Technology, #9701, 1:200), TOMM20 (Abcam, ab56783, 1:200) and Aurora B kinase (Abcam, ab2254, 1:100). Detection was mediated by incubation with AlexaFluor antibody conjugates (ThermoFisher Scientific) and nuclei were visualised using DAPI (ThermoFisher Scientific, 62248). Mounting was performed using Fluoromount-G mounting medium (ThermoFisher Scientific, 00-4958-02). For apoptotic cell death analysis, TUNEL assays (Roche, 11684795910) were performed according to the manufacturer’s instructions. Imaging was performed on a confocal microscope (Leica Sp8x, LAS X imaging software) and image analysis using ImageJ (1.51a, Java 1.8.0.231). Flow cytometry. Cells were gently dissociated (multi tissue dissociation kit, Miltenyi Biotec, 130-110-204) and incubated with LIVE/DEAD fixable green dead cell stain kit (ThermoFisher Scientific, L23101). This kit contains a fixable fluorescent dye that binds to amines. In viable cells, this amine-reactive dye binds amines on the outer cell surface, as opposed to dead cells in which the dye can additionally bind internal amines due to membrane disruption. After staining, cells were fixed (inside fix solution, Miltenyi Biotec, 130-090-477) and stained with primary antibodies (diluted in inside perm solution, Miltenyi Biotec, 130-090-477). ACTN1VioBlue (Miltenyi Biotec, 130-127-354, 1:10) and cardiac Troponin-T VioBlue (Miltenyi Biotec, 130-120-402, 1:10) were used as conjugated antibodies. As a control, universal isotype control
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