Isolation and Culture of Retinal Endothelial Cells

Retinal ECs were isolated from immorto-Cyp1b1^+/+ and -Cyp1b1^−/− mice, as previously described [14 (link)]. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, D-5523; Sigma, St. Louis, MO, USA) containing 10% fetal bovine serum (26140-079; Gibco, Grand Island, NY, USA), 2 mM L-glutamine (25030-081; Gibco), 2 mM sodium pyruvate (11360-070; Gibco), 20 mM HEPES (15630-080; Gibco), 1% non-essential amino acids (11140-050; Gibco), 100 μg/mL streptomycin, 100 U/mL penicillin (15140-122; Gibco), 55 U/mL heparin (H3149; Sigma), 100 μg/mL endothelial growth supplement at (E2759; Sigma), and murine recombinant interferon-γ (485-MI-100; R&D, Minneapolis, MN, USA) at 44 U/mL. Cells were cultured in 60 mm culture plates (12556001; Thermo Fisher, Hanover Park, IL, USA) coated with 1% gelatin (G1890; Sigma) in phosphate-buffered saline (PBS, D1408; Sigma) and maintained at 33 °C with 5% CO₂. These cells express a temperature-sensitive large T antigen, and expression is induced by interferon γ, allowing the cells to readily proliferate when cultured at 33 °C. Cells used in these studies were used before passage 15. To observe the effect of iron chelators on cellular morphologies of confluent EC layer, ~90% confluent cells were incubated with deferoxamine (DFO, D9533; Sigma) or 2,2′-Bipyridyl (Bip, D216305, Sigma), or deferiprone (DFP, 379409; Sigma) for 48 h and photographed using a phase contrast microscope (TS100; Nikon, Japan).