Culture and Transfection of Human embryonic kidney (HEK) cells
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, 2017, nontoxic}. 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.