P0 c57bl 6j

Dissociated cultures of primary cortical neurons were prepared from P0 C57BL/6J (The Jackson Laboratory) pups. All procedures were approved by Northwestern University’s Animal Care and Use Committee and were in compliance with the National Institutes of Health standards. Brains were dissected in ice-cold Leibowitz’s L-15 media with penicillin/streptomycin, and cortical tissue isolated, digested with 0.25% trypsin-EDTA solution at 37°C for 10 min, and mechanically dissociated in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 1.4 mM L-glutamine, and 6.0 g/L glucose. Cortical neurons were plated at 320,000 cells per 18 × 18 mm coverslip precoated with 50 μg/ml poly-D-lysine (Sigma) and 2 μg/ml laminin (Sigma). Neuronal cultures were maintained at 37°C in 5% CO₂ in Neurobasal media supplemented with B27 and GlutaMax-1 and penicillin/streptomycin. Neurons were transfected at day in vitro (DIV) 21 with Lipofectamine 2000 (Invitrogen). 4 μg total DNA and Lipofectamine 2000 were diluted in DMEM + HEPES (10 mM), mixed thoroughly together, and incubated for 20–30 minutes at 37°C before adding to cultured cells. Following transfection, neurons were supplanted in antibiotic-containing feeding media containing half conditioned and half fresh media, and allowed to express constructs for 4 days.

High-density cortical neuronal cultures were derived from P0 C57BL/6J (The Jackson Laboratory). They were cultured as previously described (30 , 31 (link)). All procedures involving animals were approved by the Northwestern University Animal Care and Use Committee.