Seahorse xf extracellular flux analyzer

The oxygen-consumption rates (OCR) of glioma cells and SLCs were measured using the Seahorse XF Extracellular Flux Analyzer (Seahorse Bioscience). Twenty-four-well plates (Seahorse Bioscience) were coated with 50 μL poly-d-lysine (10 μg/mL) for 2 h then coated with laminin (10 μg/mL) overnight. The next day washed the wells twice with saline, and 10,000–100,000 cells were plated per well. Three metabolic inhibitors were injected sequentially as specific time points: oligomycin (1 μM), followed by FCCP (0.75 μM), followed by a combination of rotenone and antimycinA (0.5 μM). Basal OCR were measured using the Seahorse XF24 plate reader. Several parameters of oxygen consumption were analyzed as previously reported^{25 (link)}.

The oxygen consumption rates of control and YARS cells were determined using the Seahorse XF Extracellular Flux Analyzer (Seahorse Bioscience Inc., North Billerica, MA). Briefly, cells were plated at a density of 40,000 cells/well (24-well plates (Seahorse Bioscience Inc)). The following day, the cells were washed, and fresh media were added. The sensor cartridge was loaded to dispense three metabolic inhibitors sequentially at specific time points: oligomycin (inhibitor of ATP synthase, 1 μM), followed by FCCP (a protonophore and uncoupler of mitochondrial oxidative phosphorylation, 0.5 μM), followed by the addition of a combination of R/A (mitochondrial complex I inhibitor, 1 μM). Basal oxygen consumption rate (OCR) was measured as well as the changes in oxygen consumption caused by the addition of the metabolic inhibitors described above. Several parameters were deducted from the changes in oxygen consumption, such as basal OCR and maximum mitochondrial capacity, as described previously [19 (link)].