BMDM were differentiated from bone marrow incubated in DMEM with 10% fetal calf serum (FCS), 2 mM L-glutamine, 10U/ml Penicillin, 10 μg/ml Streptomycin, and 10 ng/ml M-CSF (GenScript) for 7 days.
NIH 3T3 cells (mouse, male, ATCC CRL-1658) were maintained in DMEM supplemented with 10% Newborn Calf Serum (NBCS), 2 mM L-glutamine, 10U/ml Penicillin, and 10 μg/ml Streptomycin.
Primary corneal epithelial cells were collected from C57Bl/6 mice by incubating eyes overnight at 4 °C in a 1:1 mixture of DMEM:F12 supplemented with 4 mg/ml Dispase I (Sigma–Aldrich), 10U/ml Penicillin, 10 μg/ml Streptomycin, and 0.025 μg/ml Amphotericin B. Epithelial sheets were peeled from eyes and dissociated in TrypLE for 10 min at 37 °C with gentle agitation. Cells were passed through a 0.45 μm strainer (Corning), washed, and cultured on collagen and fibronectin-coated plates in KSFM supplemented with 5 ng/ml hEGF, 50 μg/ml Bovine Pituitary Extract, and 100 ng/ml Cholera Toxin.
All cells were maintained at 37 °C with 5% CO2. Adherent cells were detached for subculture and experiments using TrypLE. Unless otherwise noted, all cell culture materials were purchased from Gibco/Thermo Fisher.
NIH 3T3 cells (mouse, male, ATCC CRL-1658) were maintained in DMEM supplemented with 10% Newborn Calf Serum (NBCS), 2 mM L-glutamine, 10U/ml Penicillin, and 10 μg/ml Streptomycin.
Primary corneal epithelial cells were collected from C57Bl/6 mice by incubating eyes overnight at 4 °C in a 1:1 mixture of DMEM:F12 supplemented with 4 mg/ml Dispase I (Sigma–Aldrich), 10U/ml Penicillin, 10 μg/ml Streptomycin, and 0.025 μg/ml Amphotericin B. Epithelial sheets were peeled from eyes and dissociated in TrypLE for 10 min at 37 °C with gentle agitation. Cells were passed through a 0.45 μm strainer (Corning), washed, and cultured on collagen and fibronectin-coated plates in KSFM supplemented with 5 ng/ml hEGF, 50 μg/ml Bovine Pituitary Extract, and 100 ng/ml Cholera Toxin.
All cells were maintained at 37 °C with 5% CO2. Adherent cells were detached for subculture and experiments using TrypLE. Unless otherwise noted, all cell culture materials were purchased from Gibco/Thermo Fisher.