Transfection of mouse DRGs with P3 solution and program CA-137 (20µl samples) Details: Isolated DRG neurons were transfected by electroporation using a 4D-Nucleofector (Lonza, Basel, Switzerland) with the P3 Primary Cell 4D-Nucleofector X Kit S (V4XP-3032). Briefly, DRG neurons from each animal were split in two equal lots to enable experiments with wild-type and mutant NaV1.9 channels using the same batch of cells. After centrifugation (100 g for 3min) both cell pellets were resuspended individually in 20 ml of P3 primary cell solution containing supplement 1 and 0.3 mg of a plasmid encoding the enhanced green fluorescent protein. Subsequently, one lot was supplemented with 1.2 µg of a NaV1.9-encoding plasmid, while the second lot was supplemented with 1.2 µg of a vector encoding mutant NaV1.9 variants. Transfection was performed using the electroporation protocol CA137 of the 4D-Neucleofector. After electroporation, 150 ml of low calcium Roswell Park Memorial Institute (Invitrogen) 1640 medium was added to each cell suspension and cells were allowed to recover for 10 min in a 10% CO2 incubator at 37 C. The cell suspensions were then diluted in 300 ml DRG medium and immediately seeded on poly-D-lysin/laminin-coated glass coverslips, which were placed in the slots of a 24-well plate containing 1ml of DRG medium per well. DRG medium contained 89.5% DMEM/F12 (Dulbecco’s Modified Eagles Medium with Ham’s F12; Invitrogen) supplemented with 9.5% fetal calf serum and 1% penicillin/streptomycin (Invitrogen).