Extracellular acidification rate

Extracellular acidification rate (ECAR) in LLC-PK1 cells was measured using an XFp extracellular flux analyzer (Seahorse Bioscience). The cells were seeded in XFp cell culture miniplates (Seahorse Bioscience) at a density of 10⁵ cells per well in DMEM/high-glucose medium containing 10% FBS and incubated overnight. The following day, the cells were treated with 1 mM 3-AB in glucose- and serum-free DMEM medium (vehicle) for 30 minutes, and then incubated at 37℃ with XF base medium containing 4 mM glutamine in a CO₂-free incubator for 60 minutes. Glycolytic flux (basal glycolysis, glycolytic activity, and glycolytic capacity) as assessed by ECAR was analyzed by the sequential injection of 10 mM glucose, 1 mM oligomycin, and 50 mM 2-deoxyglucose. ECAR was measured at 37℃ with a 3-minute mix, 0-minute wait, and 3-minute measurement protocol. The levels of ECAR were determined three times in respective phases, and expressed as units of milli-pH (mpH) per minute.