All experimental procedures were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the guidelines were approved by the Committee on the Use and Care of Animals from the University of Fukui. As previously described,
14 RGCs from 3-day-old Sprague-Dawley rats were purified by a two-step immunopanning procedure using anti-macrophage antibodies (Accurate Chemical and Scientific Corporation, Westbury, NY, USA) and anti-Thy 1.1 antibodies derived from T11D7E2 cells (American Type Culture Collection, Manassas, VA, USA). This method is reported to yield RGCs with >99.5% purity in the literature
14 and 94.5% purity in our laboratory. RGCs were plated at a low density (2000 cells/cm
2) on 35-mm grid-lined dishes (ibidi, Martinsried, Germany) coated with poly-
d-lysine (Sigma-Aldrich Corp., St. Louis, MO, USA) and laminin (Thermo Fisher Scientific, Inc., Waltham, MA, USA). As previously described,
5 RGCs were cultured in serum-free medium (Neurobasal; Thermo Fisher Scientific, Inc.) without phenol red but with brain-derived neurotrophic factor (50 ng/mL; PeproTech, Rocky Hill, NJ, USA), ciliary neurotrophic factor (50 ng/mL; PeproTech), basic fibroblast growth factor (50 ng/mL; PeproTech), forskolin (10 μM; Sigma-Aldrich Corp.), B27 supplement (Thermo Fisher Scientific, Inc.), glutamine (Sigma-Aldrich Corp.), insulin (Sigma-Aldrich Corp.), sodium pyruvate (Thermo Fisher Scientific, Inc.), Sato Supplement, triiodothyronine (Sigma-Aldrich Corp.), and
N-acetyl-cysteine (Sigma-Aldrich Corp.). To prevent bacterial contamination, penicillin/streptomycin (Thermo Fisher Scientific, Inc.) was added to the medium. To support RGC culture, one-half of the medium was replaced at day 4 after seeding.