Measuring Cellular Respiration in SGBS Cells

Real-time oxygen consumption and extracellular acidification rates were measured by using an XF96 oximeter (Seahorse Biosciences, North Billerica, MA, USA). SGBS cells were seeded onto 96-well XF96 cell culture microplates. Cells were kept in growth medium at the longest for 24 hours and then the formerly-described differentiation process started. After recording the baseline oxygen consumption, cells received a single bolus of dibutyril-cAMP at 500 µM concentration to induce adrenergic stimulation. Then, stimulated oxygen consumption was measured in every 30 minutes. The final reading took place at 6 h post-treatment. Differentiated adipocytes were treated with 2 mM ß-guanidinopropionic acid (Sigma) to block the creatine-driven substrate cycle^{21 (link)}. In addition, proton leak respiration was measured by oligomycin (Enzo, USA) treatment at 2 µM concentration, which blocks the ATP synthase. For baseline correction, cells received a single bolus of Antimycin A (Sigma) treatment at 10 µM concentration. After the measurements, oxygen consumption rate was normalized to protein content^{27 (link),28 (link),56 (link)}.