The method to isolate RACs
15 16 was modified. Briefly, eyes from naive B10RIII or B6 mice (4–6 weeks old) were collected, and the connective tissue was removed. Then the eyes were immersed in Ca
2+/Mg
2+-free PBS containing 100 IU/mL penicillin and 100 μg/mL streptomycin on ice for 3 hours. Under a dissection microscope, the anterior segment and vitreous were discarded. The neural retina (for RACs) was incubated with 0.25% trypsin/0.53 mM EDTA at 37°C for 10 minutes Digestion was terminated by the addition of complete medium (CM; RPMI 1640 medium containing 10% fetal bovine serum, 2 mM glycine [GlutaMAX II; Gibco BRL, Life Technologies, Karlsruhe, Germany], 100 IU/mL penicillin, and 100 μg/mL streptomycin; Sigma), and the cells were centrifuged at 400
g for 5 minutes, then dissociated by gentle trituration through a fire-polished Pasteur pipette. After three washes, the cells were resuspended in CM and seeded on poly-
d-lysine–coated six-well plates at a density of 5 × 10
6 cells/well. After incubation for 30 days, the purity of the RACs was greater than 95%, as assessed by staining with primary antibody against GFAP (Sigma) and S-100 (Santa Cruz Biotechnology, Santa Cruz, CA), followed by FITC- or PE-conjugated secondary antibodies (Jackson, Pittsburgh, PA). RACs were used in experiments after culture for 3 to 5 passages.
RPE cell isolation and culture were prepared as described.
8 More than 95% of the cells in cultures stained positive with FITC-labeled anti–pan keratin antibody (clone PCK-26; Sigma-Aldrich) or RPE65 (Novus, Littleton, CO), indicating that they were virtually all RPE cells.