The generation of conditionally immortalized mouse corneal endothelial cells (MCEC) Slc4a11+/+ and Slc4a11−/− is described previously.18 (link) Cells were cultured in Complete Media, which contains OptiMEM-I medium (#51985; Thermo Fisher Scientific, Canoga Park, CA, USA), 14 mM glucose and 4 mM L-alanyl glutamine supplemented with 8% heat-inactivated fetal bovine serum (FBS; #10082139; Thermo Fisher Scientific), epidermal growth factor (EGF) 5 ng/mL (#01-107; Millipore, Darmstadt, Germany), pituitary extract 100 µg/mL (Hyclone 15 Laboratories, Logan, UT, USA), calcium chloride 200 mg/L, 0.08% chondroitin sulfate (#G6737; SigmaAldrich Corp., St. Louis, MO, USA), gentamicin 50 µg/mL (#15710072; Thermo Fisher Scientific), antibiotic/antimycotic solution diluted 1:100 (#15240062; Thermo Fisher Scientific) and 44 units/mL IFN-γ (#485-MI; R&D Systems, Minneapolis, MN, USA).
For the experiments, cells were incubated in Assay Media, which contained Earle's Balanced Salt Solution supplemented with 5.5 mM glucose (#141553; Thermo Fisher Scientific), 0.5 mM glutamine (#250030-081; Thermo Fisher Scientific), and 0.5% dialyzed FBS (#26400-036; Thermo Fisher Scientific) at 33°C for 16 hours. Drug treatments, 50 nM BafilomycinA1 (#SML1661; SigmaAldrich Corp.), 2 µM MitoQ (#317102; Medkoo Biosciences, Morrisville, NC, USA) were added into the assay media for 16 hours.
For the experiments, cells were incubated in Assay Media, which contained Earle's Balanced Salt Solution supplemented with 5.5 mM glucose (#141553; Thermo Fisher Scientific), 0.5 mM glutamine (#250030-081; Thermo Fisher Scientific), and 0.5% dialyzed FBS (#26400-036; Thermo Fisher Scientific) at 33°C for 16 hours. Drug treatments, 50 nM BafilomycinA1 (#SML1661; SigmaAldrich Corp.), 2 µM MitoQ (#317102; Medkoo Biosciences, Morrisville, NC, USA) were added into the assay media for 16 hours.
