Seahorse xf pyruvate solution

The mitochondrial respiration and glycolytic functions were measured using an XF-96e extracellular flux analyzer (Seahorse Bioscience) according to the manufacturer’s protocol. Briefly, neutrophils (2 × 10⁵ cells) were seeded in the XF assay medium and plated on XF-96e tissue culture plates precoated with Cell-Tak (Corning Inc.). The ECAR of neutrophils was measured using an XF medium containing RPMI medium supplemented with 1 mM Seahorse XF glutamine solution (Agilent Technologies), followed by sequentially injection of glucose (10 mM), oligomycin (2 μM; Sigma-Aldrich), and 2-DG (10 mM). The OCR of neutrophils was measured using an XF medium containing RPMI medium supplemented with 10 mM Seahorse XF glucose solution (Agilent Technologies), 1 mM Seahorse XF pyruvate solution (Agilent Technologies), and 1 mM Seahorse XF glutamine solution (Agilent Technologies), followed by the sequentially injection of oligomycin (1 μM), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (2 μM; Sigma-Aldrich), rotenone (1 μM; Sigma-Aldrich), and antimycin A (1 μM; Sigma-Aldrich). XF analysis was performed at intervals of 6 min over a 90-min cycle at 37 °C.

Cells were dissociated with Accutase and then suspended in high‐glucose DMEM. Cells were then seeded in an Agilent Seahorse XF24 cell culture microplate (Agilent Technologies, Santa Clara, CA, USA; 6 × 10⁴ cells per well), and the plates were incubated for 24 h. The culture medium was replaced with an analysis medium, high‐ or low‐glucose XF DMEM (Cat#103575‐100, Agilent Technologies), and the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were analyzed using an Agilent Seahorse XFe24 analyzer (Agilent Technologies). The analysis medium contained 2 mm Seahorse XF glutamine solution (Agilent Technologies), 1 mm Seahorse XF pyruvate solution (Agilent Technologies), and 25 mm glucose in the high‐glucose medium or 5.5 mm glucose in the low‐glucose medium. The OCR and ECAR were analyzed at two time points, 1 h (short period) and 24 h (long period), after replacement with fresh high‐ and low‐glucose DMEM. Additionally, the rate of ATP production was analyzed using a Seahorse XF Real‐Time ATP Rate Assay Kit (Cat# 103592‐100, Agilent Technologies), according to the manufacturer's protocol.