Assaycomplete cell plating 2 reagent

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.