Cho cells

Cell preparation: the day before the experiment, digestion of Chinese hamster ovary (CHO) cells (Sigma Chemical Co., St. Louis, MO, USA) with a density of 60–80% by trypsin, and split into some small glass plates, which placed in 35 mm petri dish, then added 10% fetal bovine serum (FBS) (Gibco, CA, USA), and Dulbecco’s modified eagle medium (DMEM) (HyClone, Logan, UT, USA) culture medium without P/S was cultured overnight in an incubator [35 (link),36 (link),37 (link)] (Supplementary Materials 1.3.1).
Electrophysiology: the cells were transferred to a perfusion tank and perfused with extracellular fluid. The intracellular fluid (mM) was: K Aspartate, 130; MgCl₂, 5; EGTA 5; HEPES, 10; Tris-ATP 4; pH 7.2 (KOH titration). The intracellular fluid was stored in small portions in a refrigerator at −80 °C and thawed on the day of the experiment. The electrodes were filled with intracellular fluid and drawn with PC-10 (Narishige, Japan). Whole-cell patch-clamp recording, noise is filtered using one-fifth of the sampling frequency. The cells were clamped at −80 mV and then depolarized to 20 mV with a square wave lasting 2 s to obtain Kv1.5 current. This procedure is repeated every 20 s. After it was stabilized, compound 1, compound 2, and acacetin were perfused, and when the reaction was stabilized, the strength of the blocking was calculated (Supplementary Materials 1.3.1).

Jurkat E 6.1 cells (Sigma Aldrich) were grown at a density of 5×10⁵ cells/ml in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS) at 37 °C in a humidified atmosphere at 5% CO2. Every 2 days the cells were split when 80% confluence was reached. CHO cells (Sigma Aldrich) were cultured in DMEM containing 10% heat-inactivated FBS. Cells were grown in culture flasks at 37 °C in a 5% CO2-humidified incubator. Cells were then allowed to grow to 70-80% confluence before transfection. Plasmid coding β3 (human) GABA_AR were transfected into CHO cells with TurboFect (ThermoFisher) transfection reagent according to the manufacturer’s protocol. Electrophysiological recordings were performed 24–48 h after transfection at room temperature.

CHO cells (Sigma) were grown using standard procedures in the following medium: Ham's F-12 Nutrient Mixture (Life Technologies), 10% fetal bovine serum (Sigma), 1% penicillin/streptomycin, 100 units/ml (Life Technologies). 24 h before transfecting cells, 35-mm dishes (Fisher) were coated with 100 μg/ml of poly-l-lysine (Sigma) and cells from a 70–80% confluent flask were trypsinized, resuspended in 5 ml of CHO medium, and a volume was taken to seed cells at a 1:10 dilution, 2 ml/dish. For transfections, an EGFP expression vector was used to enable identification of transfected cells and DNA was transfected at a ratio of 10:1, ASICx:GFP, or 5:5:1 in c-transfection experiments, using 0.9 μg of ASICx DNA and 0.09 μg of EGFP DNA (2 μg of DNA was used for nmrASIC3); the transfection reagent Lipofectamine LTX (Life Technologies) was used according to the manufacturer's protocol.

Chinese hamster ovary (CHO) cells (Sigma, passage 6 to 20) were chosen for this study due to the absence of endogenous ASIC-like currents [53] (link) and were grown using standard procedures in the following medium (CHO medium): Ham’s F-12 Nutrient Mixture (Fisher scientific, Loughborough, UK), 10% fetal bovine serum (Sigma, Gillingham, UK), 1% Penicillin/Streptomycin (100 U/ml, (Fisher scientific, Loughborough, UK). 24-hours before transfecting cells, 35 mm dishes (Fisher scientific, Loughborough, UK) were coated with 100 µg/ml poly-L-lysine (Sigma, Gillingham, UK) and cells from a 70–80% confluent flask were trypsinised, resuspended in 5 ml CHO medium and replated at a 1:10 dilution (2 ml/dish). In order to enable the identification of rASIC3-expressing CHO cells, co-transfection with a pEGFP expression vector was used together with pTRACER-rASIC3 at a ratio of 20:1 (rASIC3:GFP), using 1.5 µg rASIC3 DNA and 0.075 µg EGFP DNA; the transfection reagent Lipofectamine LTX (Invitrogen, Loughborough, UK) was used according to the manufacturer’s protocol.

CHO cells (Sigma-Aldrich) were cultured in Iscove’s medium (Biochrom) with 3.024 g/l NaHCO₃, 10% fetal bovine serum (Biochrom) and 2 mM l-glutamin (Life Technologies) at 37 °C and 5% CO₂ in humidified air. For patch-clamp experiments cells were plated in plastic dishes. After a period of 6 h, 0.5 µg plasmid DNA (of ASIC and/or P2X3) was mixed with 10 µl Polyfect® transfection reagent (Qiagen) and 100 µl OptiMEM® (Invitrogen) for 10 min. Then, the lipid–DNA complexes were introduced to the cells in 500 µl Iscove’s medium. Approximately 16 h after transfection, Iscove’s medium was replaced by OptiMEM® to remove residual plasmid DNA.

CHO cells (cat#: 85050302, Sigma-Aldrich, Vienna, Austria) were cultured at 37 °C and 5% CO₂ in Dulbecco′s Modified Eagle′s Medium/Nutrient Mixture F-12 Ham (cat#: D8437, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; cat#: F7524, Sigma-Aldrich), 100 U/mL penicillin, and 100 µg/mL streptomycin (cat#: P4333, Sigma-Aldrich). HEK 293T cells (cat#: HCL4517, Thermo Fisher Scientific, Vienna, Austria) were cultured at 37 °C and 5% CO₂ in Dulbecco′s Modified Eagle′s Medium (4.5 g/L glucose and 584 mg/L L-glutamine [cat#: L0102, Biowest, Nuaillé, France]) supplemented with 10% FBS (cat#: FBS-11A, Capricorn Scientific, Ebsdorfergrund, Germany), 100 U/mL of penicillin, and 100 µg/mL of streptomycin. Cell lines were regularly tested for mycoplasma contamination using MycoAlert Mycoplasma Detection Kit and MycoAlert Assay Control Set (cat#: LT07-418 and LT07-518, respectively, Lonza, Basel, Switzerland).