Xf96 seahorse metabolic analyzer

Ability of mitochondria in stimulated macrophages to respond to stress was measured from the Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), that were examined using the XF96 Seahorse Metabolic Analyzer from Seahorse Biosciences. Briefly, cultured BMDMs as well as immortalized macrophages were plated at a seeding density of 1 × 10⁵ cells/well in 200 μl of complete media except for the density experiments which were performed at seeding densities of 1 × 10⁵, 7.5 × 10⁴ and 5 × 10⁴ cells/well. Metabolic mitochondrial stress tests were performed via manufacturer's protocol. OCR/ECAR ratios were calculated using the difference between first three determinations of basal OCR and the last three determinations of OCR and basal ECAR where indicated.

Extracellular Acidification Rate (ECAR) for the glycolysis function and Oxygen Consumption Rate (OCR) for mitochondrial aerobic respiratory function were examined using the XF96 Seahorse Metabolic Analyzer (Seahorse Biosciences, North Billerica, MA). BMDM at day 7 of differentiation were plated at a seeding density of 0.8×10⁵ cells/well. Prior to the metabolic test, the media was removed and replaced with Seahorse XF assay media (#102365-100) containing 25 mM glucose, 2 mM glutamine, and 20 ng/mL M-CSF and the plates containing cells were incubated for 30 min at 37°C with no CO₂. For the OCR test, 1 µM Oligomycin was injected in port A, 1 µM FCCP fluoro-carbonyl cyanide phenylhydrazone in port B, and 1 µM antimycin A in port C. For the ECAR test, 56μL Glucose was injected in port A, 1 µM Oligomycin in port B, and 69μL 2-DG in port C (All reagents from Seahorse Biosciences, North Billerica, MA). Data was collected and analyzed using wave software (Wave desktop software, Agilent Technologies, China).