Cell culturing and transfection of HEK293-T for automated patch-clamp recordings

ASIC1a knockout HEK293-T cells (provided by Dr. Nina Braun; Borg et al., 2020) were grown in monolayers in T75 and T175 flasks (Orange Scientific) in cDMEM (Gibco DMEM supplemented with 10% FBS [Thermo Fisher Scientific] and 1% penicillin-streptomycin [Thermo Fisher Scientific]) and incubated at 37°C in a humidified 5% CO₂ atmosphere. Cells were split at near confluency by treatment with trypsin-EDTA (Thermo Fisher Scientific) after the cells were washed with PBS. Cells were counted with an EVE automatic cell counter (NanoEntek) and seeded into 10-cm dishes (Orange Scientific) at a density of 2.0 × 10⁶ cells/dish.

For transient transfections in 10-cm dishes, 7 µg of DNA was mixed with 21 µl of Trans-IT LT1 transfection reagent (Mirus Bio) or LipoD transfection reagent and 1,750 µl DMEM and incubated at room temperature for 20 min to allow formation of DNA–transfection reagent complexes before addition to the cells. Transfected cells were incubated for 24 h.

Transfected cells were harvested by trypsin-EDTA treatment (2 ml/10-cm dish). Detached cells were resuspended in DMEM and centrifuged at 2,000 g for 2 min. The cells were resuspended in a 1:1 mixture of DMEM and Nanion Technologies physiological solution (in mM: 140 NaCl, 4 KCl, 1 MgCl_2, 2 CaCl₂, 10 HEPES, and 5 glucose, pH 7.4). Automated patch-clamp experiments were performed 24 h after transfection.