Gibco™Neurobasal™ Medium

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Tissue isolation and neurosphere culture: Cerebral cortical tissues from mouse embryos were isolated from E12.5 C57BL/6 mice (KOATECH) or Tau-GFP mice. The dissected tissues were incubated with Accutase (Innovation Cell Technology, AT104) for 5 min at 37 °C. The dissociated cells were centrifuged at 0.2 rcf for 3 min and resuspended in a maintenance medium: DMEM/F-12 (Life Technologies, 11320033), 1% N2 (Life Technologies, 17502048), 2% B27 (Life Technologies, 17504044), and 1% penicillin/streptomycin (P/S; Life Technologies, 15140122). The cell suspension was filtered through a 40 µm cell strainer and counted using the Luna-FL automated cell counter (Logos Biosystems). The cells were seeded at 5 × 105 cells per well in an Ultra-Low Attachment 6-well Plates. Growth factors including 20 ng/ml of bFGF (R&D systems, 233-FB) and 20 ng/ml of EGF (Invitrogen, PHG0313) were added to the medium every day. For differentiation, the growth factors were removed at three days after cell seeding. On day 7 of differentiation, the neurospheres were grown in a neuronal maturation medium: 1:1 mixture of DMEM/F-12 and neurobasal medium (Life Technologies, 21103049), 0.5% N2, 2% B27, 1% P/S, and 1% GlutaMAX (Life Technologies, 35050061). From day 14 of differentiation, neuronal maturation medium containing 1 μM cyclic AMP (Sigma, D0260), 200 μM ascorbic acid (Sigma, A4403), 10 ng/ml brain-derived neurotrophic factor (BDNF, PeproTech, 450-02), and 10 ng/ml Insulin-like growth factor 1 (IGF-1; PeproTech, 250-19) were used for long-term culture. The cell medium was exchanged every 3 days.

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