OCR and ECAR were measured using an XF96 extracellular analyzer (Seahorse Bioscience). Seahorse cell plates were coated with poly-l-lysine for 40 min, washed, and then coated with or without antibodies (1 μg per well) against CD3 and CD28 overnight at 37°C. Sorted mouse iNKT cells were resuspended in medium [minimal Dulbecco’s minimum essential medium (DMEM) with 1 mM sodium pyruvate, 2 mM glutamine, and with or without 10 mM glucose, as indicated (pH 7.4)] and were seeded in the plates (5 × 105 cells per well in 180-μl medium). After centrifugation at 1500 rpm for 3 min, the plate was loaded into the instrument to determine the real-time OCR and ECAR. To measure mitochondrial respiration, cells were consecutively exposed to mitochondria-perturbing reagents, oligomycin (Oligo, 1 μM), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (1 μM), and lastly, rotenone plus antimycin A (Anti + rot, 0.5 μM). Oligomycin inhibits adenosine triphosphate (ATP) synthase and reduces mitochondrial respiration associated with ATP production, FCCP uncouples oxygen consumption from ATP production and raises oxygen consumption to a maximal value, and antimycin A plus rotenone target the electron transport chain and reduce OCR to a minimal value. The difference between OCR before oligomycin injection and OCR after rotenone plus antimycin A injection is defined as basal respiration, and difference between OCR after FCCP injection and OCR after rotenone plus antimycin A injection is defined as maximal respiration. For glycolysis stress test, medium without glucose or pyruvate was used, and glucose (Glu, 10 mM), oligomycin (Oligo, 1 μM), and 2-DG (10 mM) were consecutively added to cells. Glucose induces glycolysis, oligomycin inhibits ATP synthase and further shifts the energy production to glycolysis, and 2-DG inhibits glycolysis. The difference between ECAR after glucose injection and ECAR after 2-DG injection is defined as basal glycolysis, and difference between ECAR after oligomycin injection and ECAR after 2-DG injection is defined as maximal glycolytic capacity. To measure the OCR and ECAR of iNKT cells in different medium, iNKT cells were activated for 30 min and then were used for the measurements. To measure glucose uptake, inactivated iNKT cells and iNKT cells activated by immobilized anti-CD3 plus anti-CD28 overnight were incubated with 2-NBDG (100 μM; Invitrogen) for 10 min. To measure mitochondria activities, iNKT cells were incubated with MitoTracker Green (100 nM) for 15 min or incubated with tetramethylrhodamine (TMRM) (100 nM) for 30 min. The concentrations of lactate in culture medium were measured by lactate assay kit (Sigma-Aldrich).

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