Morphology of mouse Müller glia in growth, neurosphere, and differentiation cultures in vitro. Conditionally immortalized mouse Müller glia (ImM10 cell line) at different stages of in vitro differentiation as previously described [61
]. (a) ImM10 cells in growth media (Neurobasal, 2% fetal bovine serum, B27 supplement, and penicillin/streptomycin) under immortalizing conditions (33°C, 50 U/ml interferon gamma) show typical morphology of cultured Müller glia. (b) ImM10 cells following 4 days in sphere forming medium (Neurobasal, B27 supplement, and modified G5 supplement with 20 ng/ml EGF, 20 ng/ml FGF2, and penicillin/streptomycin) in nonimmortalizing conditions to prevent T-antigen expression (39°C, without interferon gamma), showing typical nonadherent neurospheres. (c) Spheres at 1 day following transfer to priming medium (Neurobasal, G5 supplement modified to contain EGF (20 ng/ml) but without FGF2, and penicillin/streptomycin; nonimmortalizing conditions), neurospheres adhere to plate, and cells begin to migrate onto dish. (d) Following priming, ImM10 cells in differentiation medium (Neurobasal, B27, and pen/strep; nonimmortalizing conditions) for 2 days and stained with CalceinAM show variable morphologies and include cells with distinct neuronal morphology (small cell body, multiple thin processes).