Human cadaveric donor corneas were obtained from Heartland Lions Eye Bank (St. Louis, MO, USA) within 1 week of death and primary HCEC were isolated by enzymatic digestion (Dispase II; Roche Diagnostics, Indianapolis, IN, USA) as described previously.
55 Cells were maintained in EpiLife medium with defined growth supplement (Invitrogen, Grand Island, NY, USA), 60 μM calcium chloride, and penicillin-streptomycin (5000 U/mL; 5000 μg/mL). In addition, scraped epithelial cells and freshly isolated keratocytes were collected from donor corneas and lysed for RNA collection.
56 Human telomerase–immortalized corneal epithelial cells (hTCEpi) were cultured in KGM-2 medium (Lonza, Allendale, NJ, USA)
57 and SV40-immortalized HCECs were maintained in supplemental hormonal epithelium medium (SHEM) as described previously.
58 All cells were grown to approximately 80% confluency before use in experiments. Human corneal epithelial cells were stimulated with 10
−7M vitamin D compounds (D
3 and 1,25D
3; Sigma-Aldrich Corp., St. Louis, MO, USA; 25D
3, EMD Millipore, San Diego, CA, USA), or TLR agonists (1 μg/mL Pam3CSK4 [TLR1/2], FSL1 [TLR6/2], Poly[I:C] [TLR3], Flagellin
Salmonella typhimurium [TLR5], or 10
8 cells/mL HKLM [TLR2], or 50 μg/mL zymosan [TLR2: Invivogen, Inc., San Diego, CA, USA]) for 24 hours, unless otherwise specified.