Mitochondrial Metabolism Analysis in 3T3-L1 Cells

For the analysis of mitochondrial metabolism, 3T3-L1 cells were plated in Seahorse XF cell culture plates (Agilent Technologies, Palo Alto, CA, USA) at a density of 10,000 cells per well. Cellular differentiation was induced as previously described in a prior study [53 (link)], and before measurements, the culture medium was replaced with an identical nutrient composition medium devoid of buffering agent (#103575-100, Agilent Technologies). After a 1 h incubation in a non-CO₂ incubator for degassing, the real-time oxygen consumption rate (OCR) was measured using a Seahorse XF analyzer (Agilent Technologies). Subsequently, cells were sequentially exposed to 1.5 μM of oligomycin, 1 μM of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and 1 μM of rotenone/antimycin A mixture. The OCR was monitored three times both before and after the injection of the aforementioned reagents. Mitochondrial respiration was determined by calculating the differences between the maximal OCR following FCCP injection and basal OCR. ATP production was assessed by subtracting the non-mitochondrial OCR observed after oligomycin injection from the basal OCR. The mitochondrial respiration data were adjusted to the values of the control group.