2.2. RPE cell isolation and culture

RPE cells were isolated and cultured from WT, DKO mice eyes and normal person's eyes as described previously.36, 37, 38 Using eyes provided by the Bristol and Manchester Eye Banks and after the cornea was removed for the purpose of transplantation, RPE cells were gathered and 9 human RPE cell lines, 3 from persons less than 50 years old (“young”) and 6 from over 70 year old persons (“old”), established. In brief, after removal of the anterior segment of the eye and the lens, the neuronal retinas were peeled off from the eyecups under the dissecting microscope. The RPE/choroid/sclera cups were filled with 0.5% (w/v) trypsin‐EDTA (ICN Flow, Irvine, UK) and incubated at 37°C for 1 hour. For mice, the eye was incubated for digestion after cornea and lens removal. Then, the RPE was peeled off from the choroid under microscope for cell culture and Western blotting. The RPE cells were released from the basement membrane by gentle aspiration. After two washes, single cell suspensions were cultured in a 35‐mm dish with Dulbecco's modified Eagle's medium (DMEM, Invitrogen) containing 10% (v/v) foetal calf serum (FCS, Sigma). The first passage was used for protein expression assays using Western blot. Second and third passage cells were used for measurement of trans‐epithelial electrical resistance (TEER), trans‐epithelial potential difference (TEP) and other experiments.