All cell culture reagents were obtained from Invitrogen, unless otherwise stated. Chang conjunctival cells (Wong-Kilbourne derivative) were obtained from American Type Culture Collection (CCL 20.2; Manassas, VA) and grown in M199 medium with 10% calf serum.
37 Normal human conjunctival (IOBA-NHC) epithelial cells (Diebold Y, et al.
IOVS 2002;43:ARVO E-Abstract 3170) were cultured in DMEM-F12 (1:1 vol/vol), containing 10% fetal bovine serum, 2 ng/mL mouse epidermal growth factor (EGF; Sigma-Aldrich, St. Louis, MO), 1 μg/mL bovine insulin (Sigma-Aldrich), 0.1 μg/mL cholera toxin (Sigma-Aldrich), 5 μg/mL hydrocortisone (Sigma-Aldrich), 2.5 μg/mL amphotericin B, and a penicillin streptomycin mixture (5000 U/mL and 5000 μg/mL, respectively). Human conjunctival tissue from three donors (54, 37, and 39 years of age) was obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA). Primary conjunctival epithelial cells were cultured as described by Gamache et al.
38 Briefly, conjunctival tissue was incubated overnight at 4°C in a 1:1 (vol/vol) solution of cell culture medium (EpiLife; Cascade Biologics, Portland, OR) and dispase (20 U/mL). Epithelial cells were then scraped free and seeded in the culture medium into 25 cm
2 flasks coated with a coating mix to enhance attachment (FNC; AthenaES, Baltimore, MD). The cells grew to confluence by 1 week and were then passaged in trypsin-EDTA. The epithelial nature of the primary cultured cells was studied by immunolabeling for epithelial-specific cytokeratin. All cells labeled positively for the antibody (data not shown). Primary-cultured cells of passages 1 to 3 were used for the experiments.