Rotenone antimycin a

The oxygen consumption rate (OCR) was measured by analyzing the cells using an XFp analyzer (Seahorse Bioscience, Chicopee, MA, USA). Overall, 3.5 × 10⁴ ADSC and 2.0 × 10⁴ WJ-MSC cells were cultured for 24 h after being attached to an XF cell culture miniplate pre-coated with diluted Matrigel (Corning, NY, USA, 356230). Before analysis, the medium was changed to XF Assay Medium supplemented with sodium pyruvate (Agilent, 103578-100), d-glucose (Agilent, Santa Clara, CA, USA, 103577-100), and l-glutamine (Agilent, 103579-100). To measure mitochondrial respiration, the OCR was assessed using oligomycin (1.5 µM), FCCP (0.8 µM), and rotenone/antimycin A (0.5 µM) (Agilent). The assay was performed according to the manufacturer’s instructions.

β oxidation assessment was estimated through a Seahorse Bioscience XF96 Analyzer (Seahorse Bioscience Inc., MA, USA), as described previously [39 (link)]. Briefly, oxygen consumption rate (OCR) was analyzed in FAO Assay Medium, followed by treatment with or without etomoxir (45 mmol/L). Then, 25 mmol/L XF bovine serum albumin (BSA) or 167 mmol/L XF Palmitate-BSA (#102720-100, Agilent Technologies, Wilmington, DE, USA) was added in response to oligomycin (1.2 mmol/L), fluoro-carbonyl cyanide phenylhydrazone (1 mmol/L), and rotenone/antimycin A (1 mmol/L) (#101706-100, Agilent Technologies, Wilmington, DE, USA) based on recommending processes. β oxidation was calculated according to Timper et al.’s protocol [39 (link)].

LS8 cells were plated on 96-well plates and transfected. After 48 h, cells were washed and kept in a Ringer solution containing 5 mM pyruvate and 2 mM Ca²⁺ for 2 h at 37 °C. MitoSOX Red (5 μM) (Invitrogen) and 5 μM Cell Trace Violet (Invitrogen) were loaded (30 min, room temperature) [12 (link)]. After washing with the Ringer solution, fluorescence (450/480 nm) was measured in a FlexStation 3 plate reader (Molecular Devices). The Mitochondrial Stress Test Kit (Agilent) was used to analyze mitochondrial oxygen consumption in LS8 cells, as described [12 (link),17 (link),18 (link)]. Briefly, RCAN1-transfected LS8 cells and controls (empty vector) were seeded for 24 h in an XFe24-well microplate (Agilent) at 2500 cells per well in complete DMEM (10% FBS, 1% penicillin/streptomycin, and 1% glutamine). Oligomycin (1 μM), FCCP (1 μM), and rotenone/antimycin A (0.5 μM) (Agilent) were serially added in a Seahorse XFe24 analyzer. Cell plate and compound plate were loaded into a Seahorse XFe analyzer, and OCR was analyzed. After the run, the protein content of each well was analyzed by bicinchoninic acid (BCA), and data were normalized before analyzing basal respiration, ATP production, maximal respiration, and respiratory reserve.

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using an XFp Analyzer as indicated by the manufacturer (Seahorse XF Technology, Agilent). CD8 T cells were isolated from PBMCs using a negative-selection magnetic bead kit (Miltenyi Biotec). Purified CD8 T cells were seeded at 3x10⁵ cells per well in Seahorse cell plates pre-coated with Cell-Tak (Corning). CD8 T cells were incubated for 45 min in a CO2-free incubator at 37°C before loading the plate in the Seahorse analyzer. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in XF RPMI medium supplemented with 10 mM glucose, 1 mM pyruvate and 2 mM glutamine in response to oligomycin (1 μM), FCCP (1.5 μM), rotenone/antimycin A (0.5 μM) and 2-DG (50 mM) (Agilent technologies). Maximum respiration is the average OCR values post-FCCP injection. The spare respiratory capacity (SRC) was as the OCR values post-FCCP injection minus basal OCR. Glycolysis was calculated as the basal ECAR values minus post-2-DG injection values. Glycolytic reserve was calculated as post-rotenone/antimycin A injection ECAR values minus basal ECAR values.

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.