Powerload concentrate

HiPSC‐CMs were incubated in serum‐free medium (BMCC: CaCl₂ 1.49 mM, MgSO₄*7H₂O 0.81 mM, KCl 4.4 mM, NaHCO₃ 36 mM, NaCl 77.59 mM, Na₂HO₄P 0.91 mM, Na₂SeO₃‐5H₂O 0.0001 mM, KNO₃ 0.0008 mM, d‐glucose 25 mM, sodium pyruvate 1 mM) for at least 1 h and subsequently loaded with FluoVolt Dye (1:1000, Invitrogen, Cat# F10488) and Powerload Concentrate (1:100, Invitrogen, Cat# F10488) for 20 min at 37°C. Action potentials (APs) were recorded on the CellOPTIQ system (Hortigon‐Vinagre et al. 2016 (link)) using a 40× objective, 470 nm LED, photomultiplier tube (PMT) and a sensor with an acquisition rate of 10 kHz. Plates were placed in an on‐stage incubator during the experiment, maintaining 5% CO₂ and 37°C. APs were analysed using CellOPTIQ software, which computed the time‐averaged APs (n > 3) and calculated the beating frequency, depolarization time (T_Rise) and AP duration at 90% of the amplitude (APD₉₀) (Fig. 1A).

For fluorescence spectroscopy experiments, HeLa cells (cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum) were seeded at a density of 50.000 cells per well on 24-well plates (Nunclon, Thermo Fisher Scientific) and incubated at 37ºC in a humidified atmosphere with 5% CO₂. Following 48 h incubation period, the cells were washed with PBS (pH 7.4) and incubated at 37°C for 30 min in pHrodo™ Green (Invitrogen, Thermo Fisher Scientific) staining solution (10 μl of pHrodo^TM Green AM added to 100 μl PowerLoad™ concentrate and finally diluted into 10 ml of PBS at pH 7.4). Next, the cells were washed twice and resuspended in PBS adjusted at pH 6.5, 7.4 or 8.0. After a 10 min incubation at room temperature, cells were washed twice again with PBS adjusted at the corresponding pH and fixed with 4% paraformaldehyde adjusted at pH 6.5, 7.4 or 8.0 for 10 min at room temperature (non-fixed cells were incubated in PBS adjusted at the corresponding pH). Then, the cells were washed twice again and incubated for an additional 10 min in PBS adjusted at pH 6.5, 7.4 or 8.0. Immediately afterwards, fluorescence was measured using a BioTek Synergy H1 microplate reader (Agilent Technologies) equipped with Gen5 software (bottom optics positioning, excitation: 500 nm, emission: 535 nm). All samples were prepared in triplicate.