Hek293

HEK293, HT1080, and HeLa cell lines were cultured at 37 °C and 5% CO₂ in DMEM (Dulbecco’s Modified Eagle Medium) containing high glucose (4.5 g/L; Thermo Fisher Scientific, Schwerte, Germany) with 10% fetal bovine serum (FBS) and penicillin (100 units/mL)/streptomycin (100 µg/mL). Cell lines were obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany) (HEK293: ACC 305; HeLa: ACC 57; HT1080: ACC 315).

Various cell lines, including Cos-7, HEK 293, HeLa and NIH3T3, were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), MDCK II from the American Type Culture Collection (ATCC, Manassas, USA), and wild-type murine embryonic fibroblasts (MEFs) from our laboratory. All cell lines were grown in DMEM supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Darmstadt, Germany) and penicillin/streptomycin as antibiotics. Trypsin/EDTA was from Genaxxon Bioscience GmbH, Ulm, Germany.

Cell lines HEK293 (Cat# ACC-305, RRID:CVCL_0045) and 293T (Cat# ACC-635, RRID:CVCL_0063) were purchased from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ). Regular testing for mycoplasma contamination is performed using the MycoAlert kit from Lonza. Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin and penicillin–streptomycin were purchased from Gibco (Life Technologies). DNA restriction enzymes, Phusion DNA polymerase, T4 DNA ligase and PNGase F were from New England Biolabs. Plasmid preparation kits were from Macherey-Nagel. Azi was purchased from Bachem. Protease inhibitor cOmplete EDTA-free from Roche and was used supplemented with 1 mM EDTA. Polyethylenimine MAX was from Polyscience, dissolved in H₂O as 10 mg/mL stock solution, aliquoted and stored at −20°C. 9-Fluorenylmethoxycarbonyl (Fmoc)-protected amino acids and resin were purchased from Novabiochem or Iris Biotech. Chloroacetic acid (ClAcOH) was from Sigma.

Human embryonic kidney (HEK293) cells (German Collection of microorganisms and Cell Cultures, Braunschweig, Germany; RRID:CVCL_0045) were maintained in full medium containing DMEM media supplemented with fetal bovine serum (Biochrom, Berlin, Germany), penicillin (100 U/ml, Biochrom) and streptomycin (100 μg/ml, Biochrom) with or without geneticin (G418, 100 μg/ml, Biochrom), in 5% CO₂ at 37^°C as described before (Spahn et al., 2017 (link)). Cells were passaged 1:3–1:20 every second to third day from p8 and p28 depending on confluence.
For MOR transfection, wild-type HEK293 cells were plated on 30 mm diameter plastic culture dishes coated with poly-L-lysine 24 h before transfection. Confluent HEK293 cells (70–90%) were transfected with 1 μg per 200 μl transfection mix of each plasmid containing the different cDNAs using “X-tremeGENE HP DNA Transfection Reagent” (Roche; Mannheim, Germany) following the supplier’s recommendations. The plasmid containing the cDNA encoding the FLAG-epitope-tagged rat MOR (oprm1, NM 013071.2) in pcDNA™ 3.1 vector with geneticin resistance gene was provided by Prof. Christian Zöllner (University Hamburg, Germany). The cells were later grown in T175 flasks to have enough membranes for further experiment.

Human embryonic kidney (HEK-293) cells were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and cultured in DMEM media (Biochrome, Berlin, Germany) supplemented with 4 mM L-glutamine, 10% (v/v) foetal calf serum (Biochrome) and 2 mM sodium pyruvate. Transient transfections of HEK-293 cells with the cDNAs of wild-type hTRPM2 or NvTRPM2 variants were performed using the FuGene 6 transfection reagent (Roche, Mannheim, Germany) according to the manufacturer’s instructions. In some control experiments the human NUDT9 ADPRase was co-expressed as previously described^{10 (link)}. The transfected cells were maintained for 24 h in an incubator at 37 °C and 5% CO₂. Subsequently, the cells were seeded on poly-lysine-coated glass coverslips at a suitable dilution and further incubated for 3–4 h. Then patch-clamp experiments were carried out in cells visibly positive for EGFP (and additionally DsRed in co-expression experiments). At least three independent transfections were used for each experimental group.