Maintaining and Transfecting HEK293 Cells with Nav1.6

HEK293 cells were maintained in DMEM and F-12 (Invitrogen), supplemented with 0.05% glucose, 0.5 mM pyruvate, 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen), and incubated at 37 °C with 5% CO₂. The double stable HEK293 cell line expressing CD4-Na_v1.6-C-tail-NLuc and CLuc-FGF14 was previously described^{47 (link), 48 (link)} and was maintained using selective antibiotics (0.5 mg/mL G418 and 5 μg/mL puromycin). HEK293 cells stably expressing the human Na_v1.6 channel (hereafter referred to as HEK-Na_v1.6 cells) were maintained similarly except for the addition of 500 μg/mL G418 (Invitrogen) to maintain stable Na_v1.6 expression. Cells were transfected at 80–90% confluence with equal amount (1 μg each) of plasmid pairs using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. HEK-Na_v1.6 cells were washed and re-plated at very low density prior to electrophysiological recordings.^{36 (link), 47 (link), 50 (link), 55 (link)}