2.11. Analysis of OCR and ECAR with Seahorse XF Analyser

BMDMs were seeded in Seahorse XF24 cell culture microplates (2.2 × 10⁵ cells/well) in growth medium (DMEM with high glucose and 20% FBS). For the 12-h treatment: 36 h after seeding, cells were treated with LPS (100 ng/mL) or vehicle; 11 h later, growth medium was replaced with assay medium (Seahorse XF DMEM medium supplemented with glucose (10 mM), glutamine (2 mM) and pyruvate (1 mM)) with LPS (100 ng/mL) or vehicle. Cells were incubated for an additional 60 min at 37 °C without CO₂ before a mito stress test. For the 4-h treatment: 48 h after seeding, cells were treated with LPS (100 ng/mL) or vehicle; 3 h later, growth medium was replaced with assay medium with LPS (100 ng/mL) or vehicle. Cells were incubated for an additional 60 min at 37 °C without CO₂ before a mito stress test. The mito stress test was performed in Seahorse XFe24 Analyzer (Agilent) with oligomycin (1.5 μM), FCCP (1.5 μM) and rotenone (0.5 μM) + antimycin A (0.5 μM). After analysis in Seahorse, cells were lysed with 0.1% SDS in water and analyzed for protein content with BCA protein assay. All OCR and ECAR measurements were normalized to protein content before any further analysis.