Nd7 23 cells

Culture of ND7/23 cells was performed as previously described by Zhang et al. [19 (link)]. Briefly, undifferentiated ND7/23 cells were purchased from Sigma-Aldrich (St. Louis, MO, USA) and grown in DMEM-high glucose growth media supplemented with 10% fetal bovine serum, 50 U/ml penicillin and 50 µg/ml streptomycin at 37 °C in a 5% CO₂/95% air humidified atmosphere. Cell differentiation was induced by exposing the cells to differentiation media for four to six days. The differentiation media consisted of DMEM/F12, supplemented with 0.5% fetal bovine serum, db-cAMP (1 mM), and NGF (50 ng/mL, Sigma-Aldrich, St. Louis, MO, USA).

WT or mutant SCN8A cDNA together with human β1-and β2-subunits of voltage-gated Na⁺ channels (pCLH-hβ1-EGFP and pCLH-hβ2-CD8) were transfected into ND7/23 cells (Sigma–Aldrich, RRID:CVCL_4259; cells were used at low passages, and recent mycoplasma testing has been performed) as previously described.¹⁷ (link) Electrophysiological recordings were performed 48 h after transfection and only from cells with TTX-resistant Na⁺ current that tested positive for both anti-CD8 antibody coated microbeads and green fluorescence to be sure that both β1-and β2-subunits are co-expressed. Hippocampal neurons were obtained as previously described.¹⁷ (link) WT or mutant human SCN8A cDNA (1 μg) together with cDNA encoding GFP (0.1 μg) under the promoter targeting either excitatory or inhibitory neurons (Addgene; pAAV-CAMKII-GFP, plasmid #64545; pAAV-mDlx-GFP-Fishell-1, plasmid #83900) were transfected into neurons following the standard protocol of Optifect (Invitrogen). After 48 h, electrophysiological recordings were performed from fluorescence-positive neurons. For functional investigations employing ND7/23 cells or primary hippocampal neuronal cultures, the sample size of at least three batches, each containing a minimum of three cells per batch, was determined based on previous experience to ensure reliability of the findings.

DRG neuron‐derived ND7/23 cells (Sigma Aldrich, cat. n. 92090 903) were maintained in 4.5 g/L glucose Dulbecco's Modified Eagle Medium (DMEM, Gibco, cat. n. 52100‐021, UK), 10% fetal calf serum (FCS, Sera Plus, Biotech, cat. n. 3702‐P121812, DE), 1% penicillin/streptomycin (Gibco, cat. n. 15140‐122, UK), and 0.11 g/L sodium pyruvate (Sigma‐Aldrich, cat. n. P5280, JP). The cells were cultured at 37°C and 5% CO₂ and passaged every 4 days.
Passage 8 cells were seeded at 10 000 cells/cm² for the experimental setup. Since neural differentiation of the cell line can establish a long neurite outgrowth which is similar to that observed in primary sensory neurons in culture,³² neural differentiation was induced using 1 mM N6,2′‐O‐Dibutyryladenosine3′,5′‐cyclic monophosphate sodium salt (cAMP, Sigma‐Aldrich, D0260), 10 ng/mL recombinant rat beta‐nerve growth factor (NGF, R&D, 556‐NG‐100), 0.5% FCS in IVD CM or DMEM with 1% penicillin/streptomycin, and 0.11 g/L sodium pyruvate 1 hour after cell seeding and cell attachment.

ND7/23 cells (Sigma-Aldrich, St. Louis, MO, USA) were grown on glass coverslips coated in poly-d-lysine, which were placed in 35 mm Petri dishes containing Dulbecco’s modified Eagle medium (DMEM; Biochrom GmbH) with 5% (v/v) foetal bovine serum (FBS superior; Biochrom, Berlin Germany), glutamine (Biochrom, Berlin, Germany) and 100 µg/ml penicillin/streptomycin (Biochrom, Berlin, Germany). Moreover, growth media contained 1 µM all-trans Retinal. To transiently transfect cells, 6 µl FuGENE HD transfection reagent (Promega, Madison, WI) was incubated with 2 µg of vector DNA in 250 µl DMEM for 15 min and added to the cells 2 days prior to measurements.