Seahorse xf

ATP production rate was calculated from flow-sorted CD4, CD8, and DPT using an Agilent Seahorse XF real-time ATP rate assay kit using a Seahorse XF/XFe96 analyzer and following the manufacturer’s instructions.

To directly assess the effects of MTIF3 on mitochondrial respiration in adipocytes we used the Seahorse XF (Seahorse Bioscience, North Billerica, MA) to measure cellular respiration OCR under different conditions. hWAs-iCas9 cells were seeded at 8000 cells/well in a Seahorse 24-well plate, then differentiated and induced for MTIF3 knockout or with scrambled control, as described above. Then, cells were adapted in 1 g/l growth medium (31885049, Thermo Fisher Scientific) for 3 days. Mitochondrial function was then assessed using the Seahorse XF-24 instrument according to a protocol optimized for the adipocyte cell line. Briefly, to measure OCR independent of oxidative phosphorylation, 2 μM oligomycin (O4876, Sigma-Aldrich) was added to the cells. Subsequently, 2 μM FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (C2920, Sigma-Aldrich) and 5 μM respiratory chain inhibitors: rotenone (R8875, Sigma-Aldrich) and antimycin A (A8674, Sigma-Aldrich) were added to measure maximal respiration and basal rates of non-mitochondrial respiration. Cells were then frozen at −80°C for at least 4 hr, then the plate was dried, and DNA was extracted with CyQUANT Cell Lysis Buffer (C7027, Thermo Fisher Scientific). Total DNA was then quantified by Quant-iT PicoGreen dsDNA Assay Kit (P7589, Thermo Fisher Scientific) against a lambda DNA-generated standard curve.

B12 cells were plated at a density of 40,000 cells per well in DMEM (5% FBS and 1% pen/strep) in a Seahorse XFe24 cell culture microplate and incubated overnight in the absence or presence of 100 nM DOPPA. All wells were then washed twice with bicarbonate-free Seahorse XF assay medium (Agilent) supplemented with 10 mM glucose, 4 mM L-glutamine, and 1 mM sodium pyruvate, and pH adjusted to 7.35 +/− 0.05. Following washes, the cell culture plate was incubated in the XF assay medium for 1 hour at 37 ^oC. Mitochondrial oxygen consumption rate (OCR) was first measured at baseline, and then sequentially after the administration of 1 µM oligomycin (Agilent), 1 µM FCCP (Agilent), and 0.5 µM rotenone/antimycin A (Agilent). After the Seahorse XF MitoStress Test assay, cells in control and treatment wells were lysed and protein harvested using RIPA buffer as stated earlier. Data was normalized by protein concentration.