Neuro 2a

Human embryonic kidney (HEK) 293, Neuro2a, and N1E-115 cells were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% newborn calf serum (HEK293 cells) or 10% fetal bovine serum (Neuro2a and N1E-115 cells) at 37°C. HEK293 cells were stably transfected using calcium phosphate precipitation method for transfection and 0.2 mg/ml G418 for selection. Neuro2a and N1E-115 cells were transiently transfected using jetPRIME transfection reagent (Polyplus-transfection, New York, NY, USA) and approximately 24 h later were used for ligand treatment. Cells were treated with indicated concentrations of ligands or 0.1% dimethyl sulfoxide (DMSO) as control for 24 h at 37°C. All cell culture plates were pretreated with 0.1% gelatin before cell plating unless noted otherwise.

The JEV SA-14 strain was propagated in the mosquito cell line C6/36. The supernatant containing JEV was concentrated at 100:1 and stored in aliquots at –80 °C until further use. The virus stocks were titrated by a conventional plaque assay [30 (link)].
The microglia cell line N9 and the neuroblast cell line Neuro2a were purchased from ATCC and maintained in 5% FBS DMEM and 10% FBS DMEM, respectively.

R1 murine embryonic stem cells (mESCs) were grown on 0.1% gelatin (Sigma-Aldrich) coated cell culture dishes (Invitrogen). Cells were cultured in the KnockOut^TM DMEM (Gibco) supplemented with 15% FBS (Hyclone), 1 × nonessential amino acids (Gibco), 1 × GlutaMAX^TM (Gibco), 1000 U/mL leukemia inhibitory factor (LIF) (Millipore), 100 μM 2-mercaptoethanol (Sigma-Aldrich), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). mESCs were disassociated with 0.1% trypsin (Gibco) and passaged at a split ratio of 1 to 15 every three days. R1 mESCs were kindly provided by Prof. Ye-Guang Chen at Tsinghua University. HEK 293FT cells were kindly provided by Prof. Qin Shen at Tongji University. HEK 293FT, HeLa (ATCC® CCL-2), CHO (ATCC® CCL-61), Neuro-2a (ATCC® CCL-131), HT-1080 (ATCC® CCL-121), SH-SY5Y (ATCC® CRL-2266), NCI-H1299 (ATCC® CRL-5803), A549 (ATCC® CCL-185), NIH/3T3 (ATCC® CRL-1658), MCF-7 (ATCC® HTB-22), HEK293T (ATCC® CRL-11268) cells were obtained from American Type Culture Collection and cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). Cells in all experiments were within 20 passages and free of mycoplasma contamination.

HEK293T and Neuro2a cells (both from ATCC) were maintained in DMEM with 1% penicillin/streptomycin and 10% bovine serum. Cells were solely used as protein expression systems or to produce lentivirus, and have not been authenticated or tested for mycoplasma contamination. Mouse cortical neurons were cultured from newborn mice of either sex. Cortices were dissected in ice-cold HBSS, dissociated and triturated with a siliconized pipette, and plated onto 6 mm poly l-lysine-coated coverslips (for immunofluorescence) or on 24-well plastic dishes. Plating media (MEM supplemented with 5 g/l glucose, 0.2 g/l NaHCO₃, 0.1 g/l transferrin, 0.25 g/l insulin, 0.3 g/l l-glutamine, and 10% fetal bovine serum) was replaced with growth media (MEM containing 5 g/l glucose, 0.2 g/l NaHCO₃, 0.1 g/l transferrin, 0.3 g/l l-glutamine, 5% fetal bovine serum, 2% B-27 supplement, and 2 µM cytosine arabinoside) 2 days after plating. At 6 days in vitro (DIV), neurons were transduced with recombinant lentiviruses expressing GFP-tagged, myc-tagged, or HA-tagged Munc18 variants, cre recombinase, and/or Δcre (as control). Neurons were harvested or used for experiments as indicated at 13 DIV.