Automated Patch Clamp of TRPM8-Expressing HEK293 Cells

HEK293 cells, stably expressing human TRPM8 (GenBank number NM_024080) [31 (link)], were cultured in T75 tissue culture flasks using Dulbecco’s modified Eagle medium (Euroclone, Pero, Italy), supplemented with 10% FBS (Euroclone, Pero, Italy) plus Geneticin (Sigma-Aldrich, St. Louis, MI, USA) 800 μg/mL at 37° C, in a humidified 5% CO₂ atmosphere. The cells were maintained until they reached 70~80% confluency. Prior to use, the cells were washed twice with Ca²⁺ and Mg²⁺ free PBS (Euroclone, Pero, Italy), subsequently detached using Accutase (Euroclone, Pero, Italy), and kept in suspension using CHO-SFM-II medium (Gibco, by Thermo Fisher Scientific, Carlsbad, CA, USA). The cells were then resuspended in the external solution after a centrifugation at 1000 rpm for 2 min. Single-cells suspension, with a final concentration of 3–5 million cells/mL, was required for the automated patch clamp electrophysiology procedure.