Cellular Respiratory Assay and Glycolysis Rate Measurement

An XF24 extracellular flux analyzer (Seahorse Biosciences) was applied to measure both cellular respiration and glycolysis rate according to the manufacture user guide. Macrophages were seeded on V7 microplates at a density of 500,000 cells per plate and incubated overnight. Cells were treated with either LPS (100 ng/ml) for 4 hours or IFNγ (10 ng/ml) for 16 hours. During the cellular respiratory assay, the cells were stimulated by the compounds in the following order: 2 μM oligomycin, 0.4 μM FCCP, and 4 μM antimycin A. Respiration rates were calculated as previously described³⁹. According to Seahorse XF glycolysis stress kit user guide, culture medium was replaced with glucose-free XF assay buffer containing 2 mM glutamine after 4-hour LPS (100 ng/ml) stimulation. After one-hour incubation at 37 °C under the non-CO₂ condition, cells were exposed to glycolysis stress compounds in the following order: 2 mg/ml glucose, 1 μM oligomycin, and 50 mM 2-deoxyglucose. Glycolysis rates were calculated as previously described⁴⁰.