Glucose Uptake Assay

To analyze the uptake of glucose, the fluorescent glucose analog 2(N-(7-nitrobenzen-2oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) (Cayman) was used. Also, 5 × 10⁵ neutrophils (5 × 1 0⁶/ml) were preincubated for 30 min in glucose-free XF-assay medium (SeahorseBiosciences, Denmark) supplemented with NaHCO₃ (2 g/l) or 20 nM HEPES at pH 7.4 or pH 6.0 and were then stimulated for 30 min with 20 nM PMA or left untreated. And, 10 min before the end of stimulation time, 100 mg/ml 2-NBDG was added. Following centrifugation and washing, cells were suspendend in FACS buffer and the uptake of 2-NBDG was analyzed by flow cytometry.