Xf 96 well plate

The measurement of oxygen consumption rate was carried out using Seahorse Bioscience XF-96 extracellular flux analyzer (Seahorse Bioscience). NB cells were seeded in XF 96-well plate (Seahorse Bioscience) with Seahorse XF medium supplemented with 10 mM glucose, 1 mM sodium pyruvate and 2 mM glutamine. The plate was pretreated in non-CO₂ condition for 1 h and sequentially injected oligomycin (inhibition of ATP synthase), FCCP (uncoupling agent of oxygen consumption from ATP production), and rotenone and antimycin A (inhibitor of complex I and III in electron transport chain of mitochondria) into each well, and then proceed in XF analyzer for OCR measurement and analysis.

The neutrophil extracellular acidification rate (ECAR, mpH/min) was analyzed using an XFe96 extracellular flux analyzer Glycolysis Stress Test Kit (Seahorse Bioscience, 102194-100, North Billerica, MA, USA), according to the manufacturer’s instructions. All media and injected reagents were adjusted to pH 7.4. Neutrophils were harvested, sorted and resuspended in ECAR medium (DMEM base (no bicarbonate) with 2 mM L-glutamine, 143 mM NaCl, and 0.5% phenol red (pH 7.35)) and seeded in the Seahorse XF-96-well plate coated with poly-l-lysine at a density of 0.3 × 10⁶ cells per well. Cells were starved and incubated at 37 °C in a non-CO₂ incubator for 1 h prior to the start of the assay. Three baseline ECAR measurements were made before sequential injection of glycolysis drugs. The assay consisted of four stages: (1) basal (no drugs), (2) induction of glycolysis (10 mM glucose), (3) induction of maximal glycolysis (5 µM oligomycin), and (4) inhibition of glycolysis (100 mM 2DG). Glycolytic capacity was calculated by subtracting the “mean basal ECAR values” from the “mean highest ECAR values induced by oligomycin”. At least three replicates were performed for each group.

Cells were seeded at 1.5 × 10⁴ cells/well in a Seahorse XF 96-well plate. Mito-stress test and glycolysis test were performed according to Agilent Technologies recommendations. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was used as an indicator of mitochondrial function and glycolytic activity, respectively. Analyses were performed using the Desktop Wave Software (Agilent Technologies).