Carbonyl cyanide 4 trifluoromethoxy phenylhydrazone

Oxygen consumption rate and ECAR were measured using an XF96 Extracellular Flux Analyzer (Seahorse Bioscience). Briefly, BMDMs were seeded at 8 × 10⁴ cells per well in Seahorse XF96 tissue culture plates (Agilent Technologies) and cultured overnight before treated with 5 μg/ml CTRP6 with or without 100 ng/ml LPS for 6 h. The ECAR in BMDMs were measured under basal conditions and following the addition of 20 mM glucose. Changes in oxygen consumption rate were detected under basal conditions and following the sequential addition of 2.5 μM oligomycin, 2 μM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone and 0.5 μM antimycin A/rotenone (all the chemical compounds were from Agilent Technologies). Results were collected with Wave software version 2.6 (Agilent Technologies). Data were normalized to the absorbance at 450 nm after incubation with water-soluble tetrazolium salts cell proliferation reagent (Roche, 05015944001) as a measure of cell number in each well.

CAR T-cell ECAR and OCR were measured as previously described.52 (link) XF cell culture microplate was coated with poly-D-lysine at 50 µg/mL (Sigma-Aldrich) for 24 hours at 4°C. 2×10⁵ cells per well were seeded on XF media supplemented with glucose, glutamine and pyruvate (Gibco) for 1 hour in a non-CO₂ incubator. For OCR interrogation, 1 µM of oligomycin, 1.5 µM of Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) and 1 µM of antimycin A were used (Agilent). ECAR interrogation was performed by adding 10 mM of glucose (Gibco), 1 µM of oligomycin (Agilent) and 50 mM of 2-DG (Sigma-Aldrich). ECAR and OCR were measured using Seahorse XF Analyzer (Agilent) according to the manufacturer’s instructions. For OCR parameters, non-mitochondrial respiration was subtracted. ATP-linked respiration was calculated as the mean OCR after oligomycin injection and maximal respiration as the mean OCR after FCCP injection. For ECAR parameters, non-glycolytic acidification was subtracted. Glycolysis was calculated as the mean ECAR after glucose injection and glycolytic capacity as the mean ECAR after oligomycin injection. Glycolytic reserve was calculated as the difference between glycolytic capacity and glycolysis.