Retinal Ganglion Cell Isolation and Culture

RGCs were isolated from postnatal day three Sprague-Dawley pups as described^{61 (link)} and seeded immediately in neurobasal-SATO (nb-SATO) media at 3,000 RGCs/well in 96-well plates (Falcon 087723B, Corning Inc, Corning, NY) coated with poly-D-lysine (70 kDa, 10 μg/ml, Sigma-Aldrich Corp., St. Louis, MO) and laminin (2 μg/ml, L-6274, Sigma-Aldrich Corp., St. Louis, MO). RGCs were cultured at 37 °C, 10% CO₂ for 3 days with MBV (0-80 µg/ml) or UBM-ECM (250 μg/ml). Using a Live/Dead Imaging Kit (R37601, Life Technologies, Carlsbad, CA), the number of viable RGCs per area was quantified^{14 (link)}. Five random, non-overlapping fields were imaged at 20×, totaling 45 fields of view. For total neurite growth, RGCs were labeled with anti-βIII tubulin (1:300, TUJ-1, MAB5564, Millipore, Burlington, MA). The first ten RGCs encountered, not contacting another RGC, were measured, totaling 90 RGCs per group, as described^{62 (link)}.