Purification of Primary Retinal Ganglion Cells

Primary retinal ganglion cells (RGCs) were isolated according to the two-step immunopanning method as previously reported[11 (link)]. After the deep anesthesia by isoflurane, the animals were sacrificed by CO₂ asphyxiation followed by decapitation. Then the whole retina was isolated and incubated in a papain solution (16.5U/mL, Sigma-Aldrich, US) for 30 min. Next, macrophages and endothelial cells were removed from the cell suspension by panning with an antimacrophage antiserum (Accurate Chemical, Westbury, NY). RGCs were specifically bound to the panning plates containing anti-Thy1.1 antibody (2 μg/ml; Chemicon, US) and released by trypsin treatment. RGCs were grown in serum-free basal medium (Neurobasal/B27 medium; Invitrogen, US).