Stbl2 cells

Transfections and electrophysiological experiments were performed using tsA-201 cells. Cells were grown at 37°C with 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin (50 U/mL)-streptomycin (50 μg/mL).
A plasmid encoding the major splice isoform of human SCN3A (isoform 2, which is 49 amino acids longer than isoform 1) was used (Reference Sequence NP_001075145.1), and variants were introduced by site-directed mutagenesis. Constructs were propagated in STBL2 cells at 30 °C (Invitrogen). All plasmid preparations were resequenced prior to transfection. Plasmids encoding human sodium channel auxiliary subunits β₁ (hβ₁-V5-2A-dsRed) or β₂ (pGFP-IRES- hβ2) were co-transfected in vectors containing marker genes facilitating the identification of cells expressing all three constructs (Nav1.3, Navβ₁, and Navβ₂). We performed transient transfection of 2 μg total cDNA at a ratio of Nav1.3, &beta₁, and β₂ of 10:1:1 using Lipofectamine-2000 (Invitrogen) transfection reagent. Cells were incubated for 48 hours after transfection prior to electrophysiological recording. Transfected cells were dissociated by brief exposure to trypsin/EDTA, re-suspended in supplemented DMEM medium, plated on 15 mm glass coverslips, and allowed to recover for at least 4 hours at 37 °C in 5% CO₂ prior to recording.