CCL19 is a ligand to chemokine receptor on activated macrophages (CRAM). (a) 125I-labelled CCL19 association to 106 CHO-K1 mock-transfected or CRAM-B-transfected cells with or without addition of 100 nm cold CCL19 was assessed by γ-counter. (b) Screening of several chemokines for competitive binding was carried out using 125 pm125I-labelled CCL19 and adding 25 nm CCL19 (dark grey) as a control or 100 nm for other chemokines. Potential low-affinity binding is indicated by light grey columns, no competition is shown in black, results shown are from one experiment performed in triplicate and are expressed in % of the maximal binding. (c) 125I-labelled CCL5 association to 106 CHO-K1 mock-transfected or CRAM-B-transfected cells with or without addition of 5 nm of cold CCL5 was assessed by γ-counter. (d) Competitive binding assay, with data expressed in percentage of maximum bound iodinated chemokine, determined in the absence of cold competitor. Total displacement was defined by 0·25 μm CCL19; data shown represent one representative experiment out of three, each point represents mean values and SD of triplicates and was curve fitted using a sigmoidal dose–response curve (GraphPad Prism Software) to evaluate the 50% inhibitory concentration (IC50). (e) Direct interaction of CCL19 at the indicated concentrations to Nalm6 cells was revealed by flow cytometry after addition of CCL19-biotin antibody and strepatvidin-fluorescein isothiocyanate. Data represent triplicate determinations of geometric mean fluorescence intensity from two independent evaluations and are normalized to maximal binding obtained with 100 nm CCL19.