Primary neonatal astrocyte culture

Primary astrocyte cultures were prepared from neonatal mice according to the method of Hamprecht and Löffler [46]. Briefly, as previously described [47], brains removed from newborn C57BL/6J mouse pups were mechanically dissociated by sequential passes through 250 and 135 μm nylon mesh, centrifuged at 500×g for 5 min before resuspension in DMEM supplemented with 50 U/mL penicillin-streptomycin and 10 % FBS and plated at 160,000 cells/cm². The medium was renewed every 7 days, and cultures were used between 15 and 21 days in vitro, 1–2 days after medium renewal. Immunocytochemical analysis shows these cultures contain approximately 80 % GFAP-positive astrocytes with the remainder mainly IBA1-positive microglia but no neurons (data not shown).