Uveal melanocytes were purified from human biopsy specimens, as previously described.
4 Melanoma 202 and Melanoma 285 cells were derived at the Schepens Eye Research Institute in the Ksander laboratory. The cells used are early-passage cells (less than five passages from stored frozen stock). Tumor tissue was dissected from surrounding normal uveal tissue and enzymatically digested to yield a single-cell suspension in a Petri dish containing 10 mL collagenase at 150 U/mL (Sigma-Aldrich, St. Louis, MO) in complete RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 2.0 mM glutamine (BioWhittaker), 100 U/mL penicillin, 100 U/mL streptomycin, 0.1% amphotericin B (Fungizone; BioWhittaker), 0.01 M HEPES (BioWhittaker), and 0.5% 2-β-mercaptoethanol (Sigma-Aldrich). Tissue was incubated for 90 minutes at 37°C, after which the released cells were removed and the debris allowed to settle. Cells in the culture supernatant were recovered, washed three times, and examined microscopically for the presence of viable tumor cells. Ocular melanoma cells were maintained in complete RPMI 1640 medium. Cells were incubated at 37°C with 5% CO
2 and passaged when they reached a confluent monolayer by treatment with trypsin-EDTA (BioWhittaker). Tumor cell lines were demonstrated to be ocular melanoma cells by the expression of melanoma antigen genes (MAGEs) that are transcriptionally active only in melanoma cells and are not active in normal nonmalignant cells.
5 HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), and 100 U/mL penicillin-streptomycin. Priess cells, a B lymphoblastoid cell line that expresses both class I MHC and all class II isotypes (used as a positive control in Western blot experiments) were maintained in RPMI 1640 supplemented with 10% heat-inactivated FCS and 100 U/mL penicillin-streptomycin.