| Assay Method Information | |
| | Measurement of beta-arrestin Recruitment to Human Dopamine D2L Receptors |
| Description: | PathHunter CHO-K1 cells expressing tagged human D2L receptors and tagged β-arrestin-2 (Eurofins DiscoverX, Fremont, CA, USA) were seeded into 96-well white-walled clear bottom tissue culture plates in 90 μL/cell AssayComplete™ Cell Plating 2 (CP2) reagent (Eurofins DiscoverX) at a density of 20,000 cells/well. The plates were incubated overnight in a humidified atmosphere with 5% CO2 at 37° C. Twenty to twenty-four hours later, 20 μL of test compound or vehicle in CP2 reagent and containing 2.2% DMSO was added to the cells, and they were incubated for 90 minutes at 37° C. Then, 55 μL PathHunter® Detection Reagent (Eurofins DiscoverX) was added per well and plates were incubated for 60 minutes at 25° C., followed by luminescence detection using a PHERAstar® FS multimode plate reader (BMG Labtech, Ortenberg, Germany). Raw data were converted to percent stimulation above basal values. Values were further converted to percent of maximal stimulation of β-arrestin recruitment by 30 μM dopamine. EC50 values were calculated from concentration-response curves of at least six concentrations run in duplicates by sigmoidal fitting using Origin® 7.5 software (OriginLab Corporation, Northampton, MA, USA) and were defined as the concentration of the agonist with half-maximal stimulation. The pEC50 values were calculated as the negative logarithm of the EC50 value expressed in mol/liter and is shown in Table 6. The results indicate that the examples of the present disclosure are potent agonists of the G-protein-independent signaling pathway of human recombinant D2L receptors. |
| Affinity data for this assay | |
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