Xf96 oxymeter

OC and ECAR were measured using an XF96 oxymeter (Seahorse Biosciences, North Billerica, MA, USA). hASCs of donors not related to the RNA-Seq analysis were seeded and differentiated in 96-well XF96 assay plates. Baseline, dibutyril-cAMP (Sigma-Aldrich cat#D0627) stimulated, β-guanidinopropionic acid (Sigma-Aldrich cat#G6878), and oligomycin (Enzo Life Sciences, Farmingdale, NY, USA cat#ALX-380-037) inhibited OC and ECAR were recorded. As the last step, cells received a single bolus dose of Antimycin A (Sigma-Aldrich cat# U8674) at 10 μM final concentration for baseline correction. The oxygen consumption rate (OCR) and ECAR were normalized to protein content and normalized readings were shown. For statistical analysis, relative OC and ECAR levels were determined to compare basal, cAMP stimulated and oligomycin inhibited (both in unstimulated and stimulated cells) OCRs/ECARs of each sample to the basal OCR/ECAR of untreated SC white adipocytes [6 (link),7 (link),26 (link)].

Oxygen consumption was measured using an XF96 oxymeter (Seahorse Biosciences, North Billerica, MA, USA). The hADMSCs were seeded and differentiated in 96-well XF96 assay plates. After recording the baseline OC, the cells received a single bolus of dibutyril-cAMP at 500 μm concentration modeling adrenergic stimulation. Then, stimulated OC was recorded every 30 min. The final reading took place at 5 h post treatment. To exclude that dibutyril-cAMP enhanced lipolysis might provoke artefactual mitochondrial uncoupling, we repeated the OC measurements including 2% BSA in the respiration buffer.^{44 (link)} The adipocytes were treated with 5 μm etomoxir or with 2 mm β-guanidinopropionic acid to block beta-oxidation and creatine-driven substrate cycle.^{45 (link)} Next, proton leak respiration was determined after adding oligomycin at 2 μm concentration to block ATP synthase activity. As a last step, the cells received a single bolus of Antimycin A at 10 μm concentration for baseline correction. The oxygen consumption rate (OCR) was normalized to protein content and normalized readings were displayed. For statistical analysis (n=4), the fold change of OC levels were calculated comparing the basal, cAMP-stimulated and oligomycin-inhibited (both in unstimulated and stimulated cells) OCRs of each sample to the basal OCR of untreated white adipocytes.^{38 (link), 42 (link), 46 (link)}

Oxygen consumption was measured using an XF96 oxymeter (Seahorse Biosciences, North Billerica, MA, USA) similarly to [20 (link)]. MCF-7 cells were seeded in 96-well assay plates (~2000 cell/well) and were treated for AICAR, MTX and AICAR+MTX for the time indicated. Oxygen consumption rate (OCR, reflecting mitochondrial oxidation) and changes in pH, extracellular acidification rate (ECAR, reflecting glycolysis) were recorded every 30 min to follow AICAR and MTX effect. Data were normalized to protein content and normalized readings were displayed.