Amaxa mouse neuron nucleofector kit

HEK-293 cells were transfected with pCMV-5-HT6R-EGFP and 5-HT6R-shRNA1 or scram-shRNA using TurboFect (Thermoscientific) or Lipofectamine 2000 (Life Technologies) and maintained in DMEM supplemented with 10% foetal calf serum and penicillin-streptomycin (Invitrogen) under standard conditions (37°C, 5% CO₂). NG108-15 cells were transfected using Lipofectamine 2000 (Invitrogen) with either empty plasmid or with a plasmid encoding the HA-tagged (h)5-HT6R and grown for 24 hours in DMEM supplemented with 10% foetal calf serum and HAT supplement (Life Technologies). Primary neuronal cultures were prepared as previously described (Rice et al., 2010 (link); Riccio et al., 2011 (link)). Briefly, E14.5 cortices or E17.5 cortices previously electroporated at E14.5 to label PNs were dissected in ice-cold HBSS (Life Technologies), trypsinized (0.25% Trypsin-EDTA, Life Technologies) for 20 min at 37°C and 5% CO₂, centrifuged for 5 min at 1200 rpm (124 g) and resuspended in neurobasal medium (NBM). For 5-HT6R knockdown experiments, ∼2×10⁶ cells/well were nucleofected with either 5 µg scrambled shRNA or 5 µg 5-HT6R-shRNA1, according to the Amaxa Mouse Neuron Nucleofector Kit (Lonza). Cells were seeded onto six-well plates coated with 0.25 µg/µl poly-D-lysine (Sigma) supplemented with NBM and maintained in culture at 37°C and 5% CO₂.

P7 cerebella were dissected and dispersed using the Neural Tissue Dissociation Kit P (Miltenyi) as recommended. The tissue was titurated into a single-cell suspension by using fire-polished glass Pasteur pipettes. CGNs were isolated by centrifugation over a discontinuous Percoll gradient, and the higher-density fraction, which contained 95% CGNs and 5% glia, was collected. CGNs were nucleofected with plasmid DNA by using the optimized Amaxa Mouse Neuron Nucleofector Kit (Lonza) with the O-005 program. The cells were recovered for 5 min then plated over poly-L-ornithine-, Matrigel-, laminin-, or vitronectin-coated 16-well slides or glass-bottom dishes (EMS) in Neurobasal medium supplemented with 0.5% glucose, 0.4 mg/mL of tissue culture-grade bovine serum albumin (Sigma), 2 mM l-glutamine, 50 U/mL penicillin–streptomycin, and 1× B27 supplement (ThermoFisher). When specified, insulin-free B27 supplement (ThermoFisher) was used in place of the standard B27 supplement.