Seahorse xf glucose solution

The mitochondrial respiration and glycolytic functions were measured using an XF-96e extracellular flux analyzer (Seahorse Bioscience) according to the manufacturer’s protocol. Briefly, neutrophils (2 × 10⁵ cells) were seeded in the XF assay medium and plated on XF-96e tissue culture plates precoated with Cell-Tak (Corning Inc.). The ECAR of neutrophils was measured using an XF medium containing RPMI medium supplemented with 1 mM Seahorse XF glutamine solution (Agilent Technologies), followed by sequentially injection of glucose (10 mM), oligomycin (2 μM; Sigma-Aldrich), and 2-DG (10 mM). The OCR of neutrophils was measured using an XF medium containing RPMI medium supplemented with 10 mM Seahorse XF glucose solution (Agilent Technologies), 1 mM Seahorse XF pyruvate solution (Agilent Technologies), and 1 mM Seahorse XF glutamine solution (Agilent Technologies), followed by the sequentially injection of oligomycin (1 μM), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (2 μM; Sigma-Aldrich), rotenone (1 μM; Sigma-Aldrich), and antimycin A (1 μM; Sigma-Aldrich). XF analysis was performed at intervals of 6 min over a 90-min cycle at 37 °C.

Eagle’s Minimum Essential Medium (Eagle’s MEM, Sigma-Aldrich, M0268); Dulbecco’s Modified Eagle’s Medium/Nutrient blend F-12 Ham (DMEM/F12, Caisson, DFP18); fetal bovine serum (FBS, Biowest, BIO-S1650); RPMI (Caisson, 1640); insulin (Sigma-Aldrich, I0516); penicillin/streptomycin (P/S, Caisson, PSL01); puromycin (Sigma-Aldrich, P8833); human recombinant leptin (Sigma-Aldrich, L4146); Chloroquine diphosphate salt (CQ, Sigma-Aldrich, C6628); propidium iodide (PI, Sigma-Aldrich, P4170); polybrene (hexadimetridine bromide, Sigma-Aldrich, 107689); TransIT-LT1 (Mirus, MIR2300) Seahorse XF DMEM Medium, pH 7.4 (Agilent Technologies, 103575-100); Seahorse XF glucose solution (Agilent Technologies, 103577-100); Seahorse XF sodium pyruvate solution (Agilent Technologies, 103578-100); Seahorse XF L-glutamine solution (Agilent Technologies, 103579-100); Seahorse XF calibrant solution (Agilent Technologies, 100840-000); Seahorse XF Real-Time ATP Rate Assay Kit: oligomycin, rotenone/antimycin A (Agilent, 103591-100); Seahorse XF Mito stress test kit: oligomycin, FCCP (Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone), rotenone/antimycin A (Agilent Technologies, 103015-100).