Bitan-Lab wiki: Primary neuronal culture

Primary neuronal culture

Primary cortical/hippocampal neurons were prepared from E18 pups of Sprague Dawley rat.

Reagents and accessories

Leibovitz’s L15 media, DMEM/neurobasal media, fetal bovine serum, streptomycin/penicillin (GIBCO, Invitrogen), trypsin-EDTA solution (0.25%), poly D-lysine solution, cytosine β−D arabinoside (Sigma), white/Balck plate (Costar)

Protocol

E18 pregnant rats were euthanized with CO2 and the pups were collected immediately. The cortices were dissected in chilled Leibovitz’sL15 medium in the presence of penicillin/streptomycin (1 μg/ml).

The tissue was incubated with 0.25% trypsin-EDTA solution for 30 min. and then mechanically dissociated in a small volume of Leibovitz’s L15 media using a fire-polished Pasteur pipette.

The cells were suspended in DMEM containing 10% heat inactivated fetal bovine serum and penicillin/streptomycin (1 μg/ml), and plated in poly D-lysine (0.1 mg %)-coated 96-well plates (COSTAR, Corning, USA) at a density of 3×105 cells/ml.

The cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 /95% air for 6 d before treatment with peptides.

Twenty-four hours after plating, the medium was replaced with fresh medium supplemented with 5 μM cytosine β-D-arabinofuranoside to inhibit the proliferation of glial cells.