Real time atp rate assay

The iPSM cells were dissociated with accutase for 5 min at 37 °C during the most efficient day of differentiation and re-seeded into fibronectin-coated Seahorse plates (Agilent) at a density of 7.27 x 10⁵ cells per cm² in 100 μL of Seahorse XF DMEM (Agilent) supplemented with 10 mM glucose (Agilent), 1 mM pyruvate (Agilent) and 2 mM glutamine (Agilent). Cells were allowed to attach at RT for 15 min and then transferred to a 37°C incubator without CO₂ for 40 min. After that time, 400 μL of Seahorse XF DMEM medium at 37°C were added carefully to each well without disturbing the attached cells for a total of 500 μL. Cells were incubated at 37°C without CO₂ for 15 more min. The Seahorse cartridge was hydrated overnight. For the real-time ATP rate assay (Agilent), 1 μM oligomycin, 0.5 μM rotenone and 0.5 μM antimycin A were used. All samples were run in seven to ten technical replicates in a Seahorse XFe24 (Agilent). Three biological replicates were performed for each species. The Wave Desktop and online app provided by the manufacturer were used for analysis.