Floid cell imaging station microscope system

The human embryonic kidney 293T cell line (HEK293T; American Type Culture Collection, USA) was chosen as the mammalian system for rhβ2AR overexpression. Cells were cultured in DMEM supplemented with 10% FBS, penicillin (50 U/mL) and streptomycin (50 µg/mL). Cell lines were cultivated for a minimum of 3 and a maximum of 12 sub-culturing cycles before transfection. Transfection was performed using the Turbofect transfection reagent according to the manufacturer's protocol. Transfection conditions were controlled by a means of parallel transfection with a control plasmid encoding far-red fluorescent protein mKate. Fluorescent microscope pictures of cells were obtained at 586 nm excitation and 646 nm emission wavelengths using a "FLoid" cell imaging station microscope system (Thermo Fisher Scientific, USA) to confirm the correct transfection process and correct expression of the control protein. Subsequently, transfected cell cultures underwent selection using puromycin (1 μg/mL), exploiting the puromycin resistance gene (PuroR) present in the PX459 backbone. Antibiotic selection was conducted for 21 days, thereon cells were moved to 15 cm diameter Petri dishes and grown up to ~80% confluency in 20 such dishes. Upon achieving desired confluence, cells were harvested mechanically using a cell scraper, suspended in cold PBS buffer, and centrifuged at 500×g for 5 min at 4 °C.