Extracellular Flux Analysis of Macrophage Glycolysis

The XF96 Extracellular Flux Analyzer (Agilent, Santa Clara, CA) was used to determine the glycolytic profile of cells. Oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and proton efflux rate (PER) were determined using the Glycolytic Rate Assay Kit (Agilent), according to the protocols provided by the manufacturer. Briefly, macrophages were seeded at a density of 1 x 10⁵ cells per well in the manufacture’s 96-well plate and allowed to attach overnight. Cells were then infected or not with B. abortus at a MOI of 100:1 for 24 hrs in normal cell culture medium. Prior to the assay, cells were washed tree times and media was changed to Seahorse XF DMEM medium pH 7.4 (Agilent), supplemented to contain 25 mM glucose, 4 mM L-glutamine and 2 mM pyruvate. The plate was allowed to equilibrate for 1 hr in a CO₂-free incubator at 37 °C before loading into the Seahorse analyzer. Seahorse XF96 cartridges were hydrated according to the manufacturer’s instructions. Three measurements of OCR and ECAR were performed before injection of mitochondrial inhibitors (rotenone and antimycin A) and then three additional measurements were achieved before injection of the inhibitor 2-DG. Experiments were performed with 4 replicates of each condition. The bioenergetic parameters mitoPER and glycoPER were calculated as described elsewhere [34 (link)].