Primary cortical and hippocampal neuron cultures, transfection, and infections

Primary cortical and hippocampal cultures were prepared from embryonic day 18 rat brains (of either sex). Cells were plated on coverslips coated with poly-l-lysine (37.5 μg/ml) and laminin (1.25 μg/ml) at a density of 75,000/well. Dissociated neurons were cultured in Neurobasal medium (NB) supplemented with 2% B27 (Gibco), 0.5 mm glutamine (Gibco), 15.6 μm glutamate (Sigma), and 1% penicillin/streptomycin (Gibco).

Hippocampal neurons were transfected using Lipofectamine 2000 (Invitrogen). Briefly, DNA (1.8 μg/well, for a 12-wells plate) was mixed with 3.3 μl of Lipofectamine 2000 in 200 μl of NB, incubated for 30 min, and then added to the neurons in NB at 37°C in 5% CO₂ for 45 min. Next, neurons were washed with NB and transferred to their original medium at 37°C in 5% CO₂ for 2–4 d.

Primary cortical neurons (1.3 × 10⁶ cells) were nucleofected with 0.5 μg of GFP–MACF43 or GFP plasmid and 2.5 μg of empty plasmid or shRNA using the Amaxa Rat Neuron Nucleofector kit (Lonza) according to the instructions of the manufacturer. Cells were grown for 18–24 h at 37°C in 5% CO₂ before imaging.

Hippocampal neurons were transduced with lentivirus 7–10 d before experiments. The tetracycline-dependent expression was induced 2 d before imaging by supplementing the medium with 500 ng/ml doxycycline.