2.6 |. β-Arrestin recruitment assay (PRESTO-Tango)

The PRESTO-Tango methodology was used for the screening of ligand–receptor activation using the readout of β-Arrestin recruitment as a measure. Transfected HTLA cells were starved in serum free DMEM for 2 h. Following this starvation period, cells were washed and treated with various reagents and left to incubate at 37°C, 5% CO₂ for 2 h. After this incubation period, the treatment was removed and cells were fed with fresh complete media (DMEM, supplemented with 20% FBS) and left to incubate overnight at 37°C, 5% CO₂. The following day, plates were retrieved and the growth media was replaced by the Bright-Glo^™ Luciferase Assay System reagent (E2610, Promega, Madison WI) and left to incubate for 15 min at 37°C, 5% CO₂. Luminescence was then assessed using the SpectraMax iD5 multimode microplate reader (Molecular Devices, San Jose, CA). Data are represented as fold change in luminescence using the retrieved relative fluorescence units from each sample. Treatments/reagents used to evaluate β-arrestin recruitment included the following: Vehicle ((ethanol) HBSS containing 0.01% ethanol) 20-hydroxyeicosatetraenoic acid (20-HETE) (0.0001–10 nM), N-disodiumsuccinate-20-hydroxyeicosa-6(Z),15(Z)-diencarboxamide (AAA) (1 nM), arachidonic acid (0.0001–10 nM), arachidonoyl ethanolamide (0.0001–10 nM), 12(S)-HETE (0.0001–10 nM), Vehicle (PBS), CCL5 (RANTES) in PBS (no BSA) (0.1–10 nM), BX471 (25 nM), SB328437 (80 nM) and DAPTA (20 nM). Experiments were conducted concurrently and separated for presentation clarity. The Log EC50 for all dose–response curves was generated using GraphPad Prism (ver. 8.3.0).