Assaycomplete cell culture kit 107

β-arrestin2 recruitment downstream of ACKR3 activation was analyzed using the PathHunter CHO-K1 CXCR7 β-arrestin Cell Line (93-0248C2; DiscoverX). Cells were cultured in AssayComplete™ Cell Culture Kit-107 (92-3107G; DiscoverX) at 37 °C and 5% CO₂. Briefly, 20,000 cells/well were seeded in AssayComplete™ Cell Plating 2 Reagent (93-0563R2A; DiscoverX) in a white 96-well plate (100 µL/well) with clear bottom (Costar, 3610) and incubated overnight at 37 °C and 5% CO₂. The next day, 10 µL/well of test compound (11 × concentrated) was added and cells were incubated for 90 min at 37 °C and 5% CO₂. Compounds were tested in duplicate in a 1:4 dilution series ranging from 1000 nM to 0.06 nM final concentration. After incubation, 55 µL/well of detection reagent (PathHunter Detection kit; 93-0001; DiscoverX) was added and cells were incubated for 1 h at room temperature (RT) protected from light. Finally, luminescence was measured using a FLIPR Tetra^® device (Molecular Devices, Sunnyvale, CA, USA). Four parameter non-linear curve fitting (GraphPad Prism 9.0.2) was used to determine the EC₅₀ value for β-arrestin2 recruitment.

Chinese hamster ovary CHO-K1 cells (CHO-K1) which were over expressed human D3R were cultured in assaycomplete™ cell culture kit 107 (DiscoverX, Fremont, CA, United States). Cells were seeded at a density of 25,000 cells per well of 96-well plate, and incubated at 5% CO₂, 37°C. After 46 h, HY-3-24 was (10⁻⁵ to 10⁻¹¹) in phosphate-buffered saline (PBS) was added to the cells. The plates were incubated for 30 min at 5% CO₂, 37°C for the agonist binding assay; for the antagonist assay, cells were treated with 30 nM (EC₈₀) of dopamine and then the mixture was incubated for 90 min. PathHunter™ β-arrestin recruitment assay kit (DiscoverX, Fremont, CA, United States) was added to each well, and the plate was incubated for 80 min at RT under the dark condition. The chemiluminescent signal was measured on a PerkinElmer Enspire plate reader (PerkinElmer, Boston, MA, United States). Data were analyzed by Prism followed by non-linear regression.