Rabbit Retina Tissue Culture

Pieces of rabbit retina of ca 1 cm² were placed ganglion cell side up on a 0.4 µm Millicell tissue culture insert (Millipore). The quality of the tissue after incubation depended on smooth attachment to the membrane; we found it indispensable to apply gentle suction to the tissue for ∼30 seconds during mounting. Filter stands (2 cm diameter, 1 cm high) were cut from the caps of 1 ml Monoject Tuberculin syringe jackets, so that the Millicell filter rested on four “stands” when it was placed into a 60×20 cell culture dish (Nunc). Approximately 25 ml Ames' medium (Sigma) containing 1% horse serum, 1% N2 supplement, and 100 U/ml penicillin, 100 U/ml streptomycin, 0.3 mg/ml L-glutamine (Invitrogen) were added to the dish, so that the retina was in contact with the medium via the Millicell filter over the photoreceptor side, and with the incubator atmosphere (5% CO₂, 35°C, humidified) over the ganglion cell side. All further manipulations, including gene gunning, were carried out with the retina attached to the Millicell filter. The medium was exchanged daily.