Chemotaxis assay

Assessment of mouse anti-CCR8 antibodies’ ability to block chemotaxis was done using BW5147.G.1.4 cells, a murine T lymphocyte cell line endogenously expressing CCR8. Testing was done in a 96-well Transwell plate with a 5-µm pore size in complete BW5147.G.1.4 growth medium. Cells were preincubated with test antibodies for 30 min and transferred to the top Transwell chambers in total 50 µl volume and 200,000 cells per well load. Recombinant human CCL1 (R&D Systems) was prepared at suboptimal concentration of 100 pM and added to the bottom Transwell chambers at 100 µl per well. Transwell plates were incubated at 37°C 5% CO₂ for 4 h. Ligand suboptimal concentration was established based on the cells’ chemotactic dose-response curve and varied from the effective response concentration (EC₅₀ to EC₈₀) for different experiments. At the end of incubation, the top chambers were removed and 50 µl/well CellTiterGlo reagent (Promega) was added to the bottom chambers with migrated cells. After 10 min of incubation at room temperature, 100 µl of the mix from the bottom chamber was transferred to the black well clear-bottom plates for Luminescence readout (Envision plate reader). The percent inhibition of chemotaxis was calculated using Basal and Max chemotaxis control wells present on each plate. The percent inhibition and IC₅₀ values were calculated using Screener analysis software.