RGCs were purified by a two-step immunopanning procedure, as described previously.
16 17 Briefly, the dissociated cells of retinas from 8-day-old Wister rats were incubated in flasks (Nunc A/S, Roskilde, Denmark) coated with an anti-rat macrophage monoclonal antibody (1:50; Chemicon, Temecula, CA) to exclude macrophages, and then incubated in tubes (Corning, Acton, MA) coated with an anti-rat Thy1.1 monoclonal antibody (1:300; Chemicon). RGCs adherent to the tubes were collected by centrifugation at 600 rpm for 5 minutes and seeded on 13-mm glass coverslips in a 24-well plate that had been coated with 50 μg/mL poly-
l-lysine (Sigma-Aldrich, St. Louis, MO) and 1 μg/mL laminin (Invitrogen, Carlsbad, CA). Purified RGCs were plated at a density of approximately 1000 cells/well. RGCs were cultured in serum-free B27 complete medium containing neurobasal medium
18 (Invitrogen) with 1 mM
l-glutamine (Sigma-Aldrich), B27 supplement (Invitrogen), 40 ng/mL human recombinant brain-derived neurotropic factor (BDNF; Sigma-Aldrich), 40 ng/mL rat recombinant ciliary neurotropic factor (CNTF; Peprotech, Rocky Hill, NJ), 10 μM forskolin (Sigma-Aldrich), 100 U/mL penicillin, and 100 μg/mL streptomycin. To examine the effect of acute light exposure, the cells were treated essentially as described previously.
19 Briefly, they were incubated in 1 mL of 2.5 × 10
3 mg/L ICG in PBS for 1 minute, 1 hour after their isolation, washed three times with PBS, and subjected to an acute exposure to light for 15 minutes with a standard endoillumination probe connected to a light source (Accurus; Alcon, Fort Worth, TX; spectral radiance measured as previously described
20 is shown in
Fig. 1 ). The probe was positioned at 1 cm above the cells, and the cells were irradiated evenly. The purified RGCs were then cultured for 3 days in 400 μL of serum-free B27 complete medium. To examine the effect of ICG after long exposure, RGCs were cultured for 3 days in 400 μL B27 complete medium containing ICG at a concentration of 2, 10, 25, 50, 100, and 250 mg/L. Plates were incubated in a tissue culture incubator with humidified atmosphere containing 5% CO
2 and 95% air at 37°C.