Agilent seahorse wave software

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.

The cellular OC of BAs was determined using an XFp Extracellular Flux Analyzer and analyzed by Agilent Seahorse Wave Software (Seahorse Bioscience). Prior to assay, 10,000 immortalized SVF cells were seeded into XFp microplates. One day later, Adipogenic differentiation was initiated using a protocol mentioned above. Seven days post differentiation, adipocyte culture medium was changed to XF basal medium containing 5 mM glucose, 1 mM pyruvate, and 2 mM GlutaMAX. The basal uncoupled OCR was determined using 1 μM oligomycin. To determine the impact of ISO stimulation in uncoupled OCR. Then, 5 μM ISO was injected three cycles after oligomycin injection. Oxygen consumption values were normalized to protein content.

The cellular OCR was measured using an eXF96 Extracellular Flux Analyzer and analyzed by Agilent Seahorse Wave Software (Seahorse Bioscience). Prior to assay, 2,500 primary preadipocytes were seeded into XF96 microplates. Two days after reaching full confluency, adipogenic differentiation was initiated using a protocol mentioned above. 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 hrs. Prior to 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.