2 deoxyglucose

The XF^e Extracellular Flux Analyzer (Seahorse Bioscience, Billericay, Massachusetts, USA) was employed to detect the ECAR and OCR. The ECAR was detected using the XF Glycolysis Stress Test Kit (Seahorse Bioscience), while OCR was detected using XF Cell Mito Stress Test Kit. Cells were seeded in a matched microplate at a density of 1×10⁴. In the detection of the ECAR, after performing a measurement at baseline, glucose, oligomycin (an inhibitor of oxidative phosphorylation; Selleck, Wuhan, China), and 2-deoxyglucose (an inhibitor of glycolysis; Selleck) were added to each well at specific time points. In the detection of the OCR, oligomycin, FCCP (a reversible inhibitor of oxidative phosphorylation; Selleck), and rotenone (an inhibitor of mitochondrial complex I; Selleck) plus antimycin A (an inhibitor of mitochondrial complex III; Selleck) were added in the proper order. The data were collected and analyzed using the XF-96 wave software(Seahorse Bioscience). All data were normalized based on the concentration of protein.

Bone marrow cells were differentiated for 7 d in DMEM containing 10% FBS, 20% L929 supernatant, and 1% penicillin–streptomycin at 37°C in a humidified incubator. Macrophages at day 7 were rested in DMEM containing 10% FBS and 1% penicillin–streptomycin overnight and stimulated for 24 h with or without 20 ng/ml mouse recombinant IL-4 (PeproTech) in the presence or absence of chemical inhibitors or metabolites including 1 mM 2-deoxyglucose (2-DG), 100 µM sodium iodoacetate, 500 µM sodium oxalate, 200 µM sodium oxamate, 5 µM px478 (Selleck), 200 µM D-fructose 1,6-biphosphate trisodium salt hydrate (F1,6BP), 400 µM D-3-PG disodium salt (3-PG), 5 mM sodium pyruvate (Gibco), or 1 mM L-lactate. Reagents were from Sigma unless otherwise stated.