Oxygen consumption rate analysis of MG63.3 cells

Oxygen consumption rates (OCR) were measured using a Seahorse Bioscience XF^e96 Extracellular Flux Analyzer. MG63.3 cells were plated at 10,000 cells/well in XF96 cell culture microplates. Following attachment, cells were treated with (1) vehicle, (2) 1 μM CB-839 (Selleck Chemicals), (3) 5 mM metformin (Sigma-Aldrich), or (4) 1 μM CB-839 + 5 mM metformin and incubated for 24 h at 37 °C. Just prior to the Seahorse assay, growth media was replaced with 180 μL of Seahorse XF Base Media (Agilent) supplemented with glucose, glutamine, and sodium pyruvate, and the plate incubated in a 37 °C incubator lacking CO₂ for 45 to 60 minutes. OCR was determined by performing the Cell Mito Stress Test (Agilent) according to the manufacturer’s specifications, as previously described [26 (link)].