3.8. PRESTO-Tango β-Arrestin Recruitment Assay

The HTLA cell line was used for the PRESTO-Tango β-arrestin recruitment assay [38]. The HTLA cells were provided as a gift from the laboratory of Bryan Roth (Roth Lab, USA). It is a human cell line derived from human embryonic kidney cells (HEK 293) stably expressing β-arrestin in a fusion with the tobacco etch virus protease (TEV) protease and the luciferase gene under the control of the transcription factor tetracycline transcriptional activator (tTA) [38]. The HTLA cells were cultured under standard incubation conditions (37 °C, 5% CO₂, 95% humidity) in DMEM with 10% FBS. To study the interaction of the prepared substances with the dopamine, adrenergic, and serotonin receptors, the HTLA cells were detached from the surface of the cell culture dish with 0.25% trypsin-ethylenediaminetetraacetic acid solution and seeded at the density of 1 × 10⁶ cells per culture dish with the diameter of 60 mm in 5 mL of DMEM supplemented with 10% FBS. After 24 h of cultivation, the cells were transfected with 2 µg of plasmid DNA using JetOPTIMUS transfection reagent (Polyplus, Berkeley, CA, USA), according to the manufacturer’s protocol.

The transfection was performed by using plasmid DNA for individual types of the dopamine, adrenergic, and serotonin receptors in a fusion with the Tango construct (Table S3).

The transfected HTLA cells were incubated for 24 h, and then 1 × 10⁴ cells were seeded per well of a white 96-well clear bottom plate (Brand, Germany) in 40 μL of DMEM with 1% charcoal-stripped FBS (Thermo Fisher Scientific, USA). After 6 h of incubation, the cells were treated with 5× concentrated test substance solution (10 µL per well) diluted in DMEM with 1% charcoal-stripped FBS. The final concentrations of the solutions in the wells were 0.5, 1, 5, 10, 50, 100, and 500 nM. Transfected cells treated with dopamine, serotonin, and noradrenaline as well as untreated (transfected) cells served as controls. After the treatment, the cells were further cultured for 20 h under standard cultivation conditions. Then, 50 μL of pre-prepared OneGlo reagent (Promega, Madison, WI, USA) containing a luciferase substrate and lysis reagent was added to the wells. After 5 min., the luminescence was measured using a multi-mode microplate reader SpectraMax iD5 (Molecular Devices, San Jose, CA, USA) measuring the entire wavelength spectrum. All samples were measured in quadruplicates. The data were evaluated by Prism 7 software (GraphPad, San Jose, CA, USA).