Mouse neuronal N1E-115 and green monkey kidney epithelial COS-7 cells (JCRB Cell Bank, Osaka, Japan, and Japan Health Sciences Foundation, Osaka, Japan) were cultured on 6- or 10-cm cell culture dishes (ThermoFisher Scientific, Waltham, MA, USA) in high-glucose Dulbecco’s modified Eagle medium (DMEM; Nacalai Tesque) containing 10% heat-inactivated fetal bovine serum (FBS; ThermoFisher Scientific) and penicillin-streptomycin solution (Nacalai Tesque) in 5% CO2 at 37 °C.
COS-7 and N1E-115 cells are high transfection efficiency and neuronal differentiation abilities, respectively [13 (link),14 (link)]. Cell lines stably expressing the wild-type (indicated as WT in the figures) Chmp2b gene or the gene with the T104N mutation were selected in the presence of G418 (Nacalai Tesque) as described previously [15 (link)] and isolated as a single clone. To induce differentiation, N1E-115 cells were cultured in DMEM and 1% FBS containing penicillin-streptomycin in 5% CO2 at 37 °C for 48 h. Cells with processes longer than their cell body length were considered to be neurite-bearing cells (i.e., differentiated cells) [16 (link)]. Under these conditions, in each experiment, it was estimated that less than 5% of adherent cells incorporated trypan blue (Nacalai Tesque).
COS-7 and N1E-115 cells are high transfection efficiency and neuronal differentiation abilities, respectively [13 (link),14 (link)]. Cell lines stably expressing the wild-type (indicated as WT in the figures) Chmp2b gene or the gene with the T104N mutation were selected in the presence of G418 (Nacalai Tesque) as described previously [15 (link)] and isolated as a single clone. To induce differentiation, N1E-115 cells were cultured in DMEM and 1% FBS containing penicillin-streptomycin in 5% CO2 at 37 °C for 48 h. Cells with processes longer than their cell body length were considered to be neurite-bearing cells (i.e., differentiated cells) [16 (link)]. Under these conditions, in each experiment, it was estimated that less than 5% of adherent cells incorporated trypan blue (Nacalai Tesque).
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