Metabolic Extracellular Flux Analysis

Metabolic extracellular flux was measured using Seahorse Extracellular Flux Analyzer XF-24 (Agilent Technology) as previously described^{90 (link)}. Briefly, 1.7 × 10⁵ MB or hNSC cells were seeded on XF-24 cell culture microplates, coated with 3.4 mg/mL BD Cell-TakTM tissue adhesive solution (BD Bioscience 354240), on the same day of the analysis. For OCR and ECAR analysis after IP6 treatment, cells were incubated with IP6 for 24 h before seeding on coated wells. Microplates containing the cell suspension were centrifuged at 180×g for 2 min and then incubated at 37 °C with unbuffered DMEM (Sigma) in a non-CO₂ incubator for at least 2 h. OCR was determined following sequential treatment with the ATPase inhibitor oligomycin (5 µM), the uncoupling agent FCCP (4 µM) and the electron-transport-chain inhibitors rotenone (10 µM) and antimycin A (10 µM). ECAR was measured in response to glucose (10 mM), oligomycin (5 µM) and 2DG (50 mM) treatment.