GTPγS assay

The GTPγS assay format has been described previously for other chemokine receptor targets.⁴² Briefly, CHO-CCR1 cell membranes (5 µg/ml) were mixed at a 1:1 ratio with 25 mg/ml WGA-coupled PS imaging (Leadseeker) beads (Perkin-Elmer) before being incubated for 1 h at 4°C. GDP was added to 384-well solid white plates (Nunc, FAC 4.4 μM) containing test compound. ³⁵S-GTPγS (Perkin-Elmer) was diluted 1:1200 in assay buffer and 20 μl/well added to the plates before centrifugation at 1200 rpm for 30 s. After 3 h plates were read using Viewlux (Perkin-Elmer) with a 613/55(A09) emission filter. The raw data was analyzed using a 4-parameter logistic fit IC₅₀ template (XLFit).