Assay was performed as previously described (65). An XCL1 tracer was produced by iodine labeling of tyrosine residues of the XCL1 protein by using an oxidative iodination procedure [using chloramine-T to incorporate a 125I isotope (PerkinElmer)], and the product was purified and verified by reversed-phase high-performance liquid chromatography (HPLC) (66). COS-7 cells transfected with XCR1 were seeded in 96-well plates at 35,000 cells per well (in duplicates) for growth 1 day before the assay. On the day of the assay, the cells were washed and changed to a 50 mM Hepes buffer supplemented with bovine serum albumin (BSA) (5 g/liter) and chilled to 5°C. Ligands were added shortly before the labeled XCL1 CC3 tracer (calibrated to result in ≈10% tracer binding), and the cells were incubated at 4°C for 3 hours before being washed in a 50 mM Hepes buffer containing BSA (5 g/liter) and NaCl (29.22 g/liter). The cells were lysed, and γ radiation of the lysate was measured.

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