2.5. Oxygen Consumption Rates (OCR) and Extracellular Acidification Rates (ECAR)

Macrophages were seeded at 1 × 10⁵ cells/well in 24 well plates designed for the Seahorse^® XF24 Extracellular Flux Analyzer (Agilent, Santa Clara, CA, USA). The dissolved oxygen concentration in the extracellular medium was evaluated, providing the cellular oxygen consumption rate (OCR) and the changes in the pH reflecting the rate of glycolysis. Twenty-four hours after cell seeding, the medium was changed and cells were treated as described below. One hour before the assay, cells were washed with RPMI medium without fetal bovine serum, sodium bicarbonate and phenol red, at 37 °C in a CO₂-free atmosphere. Then, different drugs that modulate mitochondrial respiration rates were sequentially injected into the medium: oligomycin 1 µM (ATP synthase inhibitor), FCCP 1.25 µM (mitochondrial uncoupler), and a mix of antimycin A 1 µM (complex 3 inhibitor) and rotenone 1 µM (complex 1 inhibitor). The OCR measured in each condition were calculated as follows: 1. Non-mitochondrial oxygen consumption: The minimum rate measurement after rotenone and antimycin injection; 2. Basal respiration: (last rate measurement before the oligomycin injection) minus (non-mitochondrial oxygen consumption rate); 3. Maximal respiration: (maximal rate measurement after FCCP injection) minus (non-mitochondrial oxygen consumption rate); 4. Proton leak: (minimal rate measurement after oligomycin injection) minus (non-mitochondrial oxygen consumption rate); 5. ATP-linked respiration: (last rate measurement before the oligomycin injection) minus (minimal rate measurement after oligomycin injection); 6. Spare respiratory capacity: (maximal respiration) minus (basal respiration). The medium acidification rates calculations were: 1. Glycolysis: Last rate measurement prior to oligomycin injection; 2. Glycolytic reserve: (maximum measurement rate after oligomycin injection) minus (last rate measurement prior to oligomycin injection); 3. Glycolysis Capacity: (Glycolysis) plus (Glycolytic reserve).