Exf96 extracellular flux analyzer

The Extracellular Acidification Rate (ECAR) was measured using a Glycolysis Stress Test Kit and eXF96 Extracellular Flux Analyzer (Seahorse Bioscience) according to the manufacturer’s protocol. In brief, 10,000 cells were plated in 100 μl of their standard growing media and cultured overnight. On the day of the measurement, cells were washed with XF media and incubated in a CO2-free incubator at 37°C for 2 h to establish equilibration before loading. ECAR measurements were taken before and after the addition of glucose (10 mM), oligomycin (1 mM) and 2-DG (50 mM) and used to calculate glycolysis, glycolytic capacity and glycolytic reserve.

The cellular OCR was measured using an eXF96 Extracellular Flux Analyzer and analyzed by Agilent Seahorse Wave Software (Seahorse Bioscience). Before assay, 2,500 primary preadipocytes were seeded and differentiated in XF96 microplates. Once fully differentiated, adipocyte culture medium was changed to assay medium containing 25 mM glucose, 1 mM pyruvate and 2 mM l-glutamine and 0.5 mM carnitine without phenol red or sodium bicarbonate for 3 h. Before the measurement, cells were incubated in a CO₂-free incubator for 15 min. Basal rates of respiration were measured in assay medium and followed with sequential injections of oligomycin (2 μM), FCCP (0.5 μM) and rotenone with antimycin A (each 0.5 μM). Oxygen consumption values were normalized to protein content.

An eXF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA) was used to determine the effects of the drugs on lactate production (culture medium acidification) in T47 Ctrl and PTEN KO cells. Cells were seeded at 40,000 cells/well into 96-well Seahorse culture plates and treated with or without drugs for 72 h. Cells were then incubated for 1 hour at 37°C after replacement of the medium with Seahorse incubation medium containing 10 mM glucose, 2 mM L-glutamine and 5 mM HEPES (pH 7.4), with or without drugs. Incubations were performed in a CO₂-free incubator to ensure accurate measurements of extracellular pH. Measurements of total extracellular acidification rate (ECAR, sum of glycolytic acidification, in the form of lactate^- plus H⁺ and the respiratory acidification, in the form of CO₂ (which hydrates to H₂CO₃ then dissociates to HCO₃^- plus H⁺)) were performed according to the manufacturer’s instructions. ECAR is presented as mean ± standard deviation (S.D.) of experimental quintuplicates.