Xf media

Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured using an XFe24 Extracellular Flux Analyzer (Seahorse Bioscience). Before analysis, human CD4⁺ T cells enriched from PBMC and murine Th17 cells differentiated from naive CD4⁺ T cells were counted. OCR was measured using the mitochondria stress test protocol. Cells were resuspended in XF media (Seahorse Biosciences) supplemented with 10 mM glucose (Sigma‐Aldrich), 1 mM GlutaMAX (GIBCO), and 1 mM sodium pyruvate (Corning). OCR was measured under basal conditions and upon treatment with the following reagents: the ATP synthase inhibitor oligomycin (1 μM); the protonophore carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone (FCCP) (1.5 μM) to uncouple mitochondria; the mitochondrial complex I inhibitor rotenone (100 nM); and the mitochondrial complex III inhibitor antimycin A (1 μM). For ECAR measurements, cells were resuspended in XF media (Seahorse Biosciences) supplemented with 1 mM GlutaMAX (GIBCO) and 1 mM sodium pyruvate (Corning). ECAR was measured under basal conditions and upon addition of the following reagents: glucose (25 mM); oligomycin (5 μM); and 2‐DG (100 mM) to block glycolysis. Different OXPHOS and glycolysis parameters were calculated as described previously (Vaeth et al, 2017a).

Oxygen consumption rate (OCR) was measured using Seahorse XF96 Analyzers (Agilent, Santa Clara, CA, USA) according to the manufacturer’s protocol. Prior to the start of the experiment, cells were seeded (8000 cells/well) into the XF96 cell culture plate and allowed to adhere for 24 h. Cell culture media were replaced with XF media (Seahorse Bioscience, North Billerica, MA, USA) supplemented with 2 mmol/L Glutamax, 1 mmol/L sodium pyruvate, and 25 mmol/L glucose and were incubated for 1 h in a 37 °C CO₂ incubator. Following the establishment of a basal OCR reading, the following pharmacological manipulators of mitochondrial respiratory chain proteins were injected sequentially: (i) oligomycin (1 μmol/L) at ATP synthase inhibitor; (ii) carbonyl cyanide-4-(trifluoromethoxy) phenyl hydrazone (FCCP) (1.5 μmol/L), as a mitochondrial uncoupler; followed by (iii) antimycin A (10 μmol/L), as a complex III inhibitor. The results were calculated using the Seahorse XF Mito Stress Test Report Generator (Seahorse Bioscience, North Billerica, MA, USA).