Rat cortical astrocytes

To aid in iNGN survival [17 (link)] and synapse formation [18 (link)], astrocytes were co-cultured with iNGNs. Rat cortical astrocytes (Life Technologies) were routinely cultured in astrocyte medium (ACM = DMEM + 15% fetal bovine serum + N2 supplement; Sigma-Aldrich, Sigma,-Aldrich, and Life Technologies, respectively) containing penicillin/streptomycin (100 units penicillin and 100 μg streptomycin/ml final concentration; Sigma-Aldrich), and passaged using StemPro Accutase (Life Technologies). Astrocytes were passaged a minimum of 3 times before co-culture, to ensure there were no neurons, and used until a maximum of passage 12. One to three days before co-culture, 2.5x10⁴ astrocytes were seeded onto coverslips coated with 100 μg/ml PDL, in 24-well plates (Nunc/Thermo Fisher Scientific).

Ganglionic eminence and subventricular zone were isolated from embryonic wt mice, adult wt and p53 null mice, respectively. Tissues were mechanically dissociated using a P-1000 pipette. Cells were then plated at a density roughly of 500000-1000000 cells in Corning^® Ultra-Low attachment cell culture flasks 25 or 75 cm² in Stemcell Technologies NeuroCult^® NS-A Proliferation Kit (Mouse) supplemented with 2 μg/ml Heparin, EGF 20 ng/ml and bFGF 20 ng/ml. Cells were continuously given new media and growth factors weekly and passaged every one to two weeks dependent on growth rate. Cell differentiation was performed by plating cells in Stemcell Technologies NeuroCult^® NS-A Differentiation Kit (Mouse) on Laminin-1 (Sigma) coated (100 μg/ml Laminin-1/PBS for 1 hour at 37 °C) glass slides (22×22 mm) in 6-well plates. Rat cortical astrocytes (Life technologies) were grown as adherent monolayer culture in DMEM with 10% FBS and thereafter fixed and processed for IF.