Xf96 oximeter

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)}.

OCR and ECAR were measured by XF96 oximeter (Seahorse Biosciences, North Billerica, MA, USA). Cells were seeded and differentiated on XF96 assay plates with or without BMP7. During measurement, baseline respiration and acidification were measured for 30 min followed by stimulated OCR and ECAR. A single bolus dose of dibutyryl-cAMP (at 500 μM final concentration) was added to mimic adrenergic stimulation leading to the stimulated OCR and ECAR, which were measured at 30-min intervals. 3 h post treatment, oligomycin at 2 μM final concentration was added to block ATP synthase activity to measure proton leak OCR. For measuring creatine cycle related OCR, β-GPA was added at 2 mM concentration after cAMP treatment. Antimycin A at 10 μM final concentration was added at the end for baseline correction. The OCR was normalized to protein content [40 (link)].

Oxygen consumption was measured using an XF96 oximeter (Seahorse Biosciences, North Billerica, MA, USA). Cells were seeded and differentiated in 96-well XF96 assay plates. On the day of measurement, after recording the baseline oxygen consumption, cells received a single bolus dose of dibutyril-cAMP (500 μM final concentration) simulating adrenergic stimulation. Then, stimulated oxygen consumption was recorded every 30 minutes. The final reading took place at 7 h post-treatment. As a last step, cells received a single bolus dose of antimycin A (10 μM) for baseline correction [14 (link), 34 ].

Cells were seeded and differentiated on XF96 assay plates (Seahorse Biosciences, North Billerica, MA, United States) to white, active beige, or inactive beige using the protocols as described in Section 2.3. The OCR and ECAR were measured with XF96 oximeter (Seahorse Biosciences). Dibutyryl-cAMP stimulated OCR and ECAR, etomoxir-resistant (ETO-R) OCR, and stimulated proton leak OCR were quantified using previously utilized protocols (Kristóf et al., 2015 (link); Arianti et al., 2021 (link); Nagy et al., 2022 (link)). 10 μM antimycin A was used for baseline collection (measuring non-mitochondrial respiration). The OCR was normalized to protein content of each well.