Binding Competition Assay

The assay was performed as previously described.^27,45 In brief, HEK-ACKR3 cells were distributed into 96-well plates (2 × 10⁵ cells/well) and incubated with a mixture of CXCL12–AF647 (5 nM) and unlabeled peptides at indicated concentrations in FACS buffer (PBS, 1% BSA, 0.1% NaN₃) for 90 min on ice. After two washing steps, the cells were incubated for 30 min at 4 °C with Zombie Green viability dye (BioLegend). After two washing steps, the cells were resuspended in FACS buffer and mean fluorescence intensity (MFI) was measured from 10 000 gated cells using a BD LSR Fortessa flow cytometer. The signal obtained for CXCL12–AF647 in the absence of unlabeled ligands was defined as 100% binding, and signal for CXCL12–AF647 in the presence of 1 μM unlabeled CXCL12 was set to 0%.