All procedures involving mice were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care and Use Committee at the University of Miami. Retinas from wild-type (wt) postnatal day (P) 8 mice were dissected free of choroidal vessels and dissociated by activated papain for 30 minutes at 37°C, using protocols modified from Barres et al.
24 and Wahlin et al.
25 Neurobasal medium containing 1× lo ovomucoid (LoOvo) plus DNAse I (Invitrogen, Carlsbad, CA) was added, and the cells were centrifuged at 800 rpm for 8 minutes at room temperature. The cell pellet was resuspended in neurobasal-LoOvo medium without DNAse, recentrifuged, and resuspended in neurobasal medium containing
l-glutamine, B27, and antibiotics. Cells were plated onto poly-
d-lysine/laminin–coated 96-well dishes at 2.5 × 10
5 cells per well. Cultures with fibroblast or endothelial growth, which appeared as large, flat cells, were excluded. Immunostaining with cell type marker antibodies demonstrated that the retinal cultures were almost entirely composed of Müller glia and photoreceptors (see Results). Antibodies against the following cell-type marker proteins were used: calbindin (horizontal cells and a subset of amacrine cells), 1:500 dilution (Novus Biologicals, Littleton, CO), glutamine synthetase (Müller glia), 1:300 dilution (Sigma, St. Louis, MO), Pax6 (amacrine, ganglion), 1:100 dilution (Santa Cruz Biotechnology, Santa Cruz, CA), vimentin (astrocytes and Müller glia), 1:200 (Sigma), rhodopsin (rod photoreceptors), 1:300 (Chemicon, Temecula, CA), PKCα (rod bipolar), 1:200 (Novus Biologicals), and Thy1 (ganglion cells) 1:100 (Santa Cruz Biotechnology).