All procedures using animals adhered to the Association for Research in Vision and Ophthalmology statement for the use of animals and the NEI's Institutional Animal Care and Use Committee approved protocols. Mouse RPE was isolated from retinas of C57/B6J mice at 6–8 weeks of age as described previously [15 (link)]. Briefly, mice were euthanized, and their eyes were enucleated. The globes were washed with PBS containing 1% penicillin-streptomycin (Sigma) and then were dissected free of periocular connective tissue. Then, the globe was placed on 2% Dispase II (neutral protease, grade II, Roche, Indianapolis, IN, USA) and incubated at 37°C for 40 min. The globe was transferred to DMEM/F12 media, the anterior segment was removed, and the retina containing the RPE layer was dissected free. The loosely adherent RPE cell layer was gently separated from the retina and transferred to a 15 mL tube containing DMEM/F12, 20% FBS, and 1% L-glutamine-penicillin-streptomycin. Cells were then centrifuged at 1000 rpm for 5 min and resuspended. The RPE suspension was added to 6-well cell culture plates. The medium was changed after 5-6 days and every 2-3 days thereafter. The RPE cells between two and three passages were stimulated with 100 ng/mL recombinant mouse IL-1β (R&D Systems), 10 ng/mL recombinant mouse IL-18 (MBL), or 10 ng/mL recombinant mouse IL-17 (R&D Systems) for 24 hours.
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