Cortical astrocyte cultures were prepared from P2 mouse pups60 of mixed sex. All animal protocols were conducted according to national guidelines approved by the Johns Hopkins Animal Care and Use Committee. After dissection and removal of the meninges and blood vessels, cortices were incubated with trypsin-EDTA (0.05%, 0.2 mm) for 20 min at 37°C. Tissue was triturated and suspended in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 10% Hams F-12, and 0.24% penicillin/streptomycin (10,000 U/ml penicillin, 10,000 mg/ml streptomycin). Cells (14 ml) were plated at a density of 2.5 × 105 cells/ml (3 × 104 cells/cm2) in 75 cm2 flasks and maintained in a 5% CO2 incubator at 37°C. The growth medium was completely exchanged with fresh medium twice a week until cells were 90% confluent (9–10 d).
