4.9.2. Seahorse XF Glycolysis Stress Test Assay

Neutrophils were suspended in PBS (glycolysis stress test medium, w/o glucose and pyruvate supplementation, pH 7.4), plated (300,000 cells/well) in 160 μL on Agilent Seahorse 8-well XFp Cell Culture Miniplate and allowed to adhere for 30 min at 37 °C. Just before running the assay, neutrophils were stimulated with LPS (75 µg/mL final well conc.) and immediately placed in the instrument. The assay workflow was as follows: (1) injection of glucose (10 mM; rate of glycolysis), (2) injection of ATP synthase inhibitor - oligomycin (1 µM; cellular maximum glycolytic capacity). (3) Lastly, 2-DG (50 mM) was injected to abolish glycolysis. Measurements after each injection consisted of a mixing time of 3 min each, and a data acquisition period of 18 min consisting of 3 cycles. The difference between glycolytic capacity and glycolysis rate defines glycolytic reserve. Three cycles of baseline measurement (total duration of 18 min) were taken prior to the addition of glucose. ECAR, prior to glucose injection, is referred to as nonglycolytic acidification. All parameters were calculated using Agilent Seahorse XF Glycolysis Stress Test Report Generator.