Unbuffered dmem

To study the effect of NO on mitochondrial energetics, O₂ consumption rate (OCR) and extracellular acidification rate (ECAR) were studied with the Seahorse XF24 analyzer (Seahorse Bioscience, Massachusetts, USA). HS181 cells were seeded onto Matrigel-coated Seahorse plates. After 48 h, cells were washed 3 times with 0.9 Na Cl %. Subsequently, 675 µL of Seahorse medium (unbuffered DMEM, Sigma) was added. Plates were pretreated for 1 h at 37 °C in CO₂-free conditions to reach temperature and pH equilibrium. Then, port 1 was loaded with basal medium, port 2 was loaded with oligomycin at a final concentration of 6 µM, port 3 was loaded with FCCP (0.3 µM) and port 4 was loaded with rotenone and antimycin (0.1 μM). The mitochondrial stress protocol started by measuring baseline oxygen consumption rate (OCR) followed by measurement of OCR changes in response to injection of oligomycin, FCCP, and finally antimycin and rotenone. Oxygen consumption and ECAR were measured with the Seahorse. Basal respiration was defined as the average values measured from time point 1 to 5 (0–45 min). ATP production, proton leak, and non-mitochondrial respiration were calculated according to Seahorse XF cell mito stress parameter equations. Data were normalized to the amount of protein present in each well, which was quantified by BCA (Pierce BCA Protein Assay Kit (Thermo Fisher Scientific)).

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.