(A) CCR1 is a receptor for CKβ8-1 and CKβ8. HOS cells (2 × 10⁷) expressing CCR1 were loaded with fura-2AM at 5 μmol/L for 30 minutes and washed. Cells (2 × 10⁶) in 2 mL were used for Ca²⁺ flux assay. For this assay, 25 nmol/L of each chemokine was used. CCR1 expressing HOS cells were treated with 25 nmol/L of IL-8, MIP-1α, and MIP-1α, successively. Their Ca²⁺ response was measured. For desensitization experiments, the HOS cells were treated with 25 nmol/L of MIP-1α and CKβ8-1 or CKβ8-1 and MIP-1α, or were treated with 25 nmol/L of MIP-1α and CKβ8 or CKβ8 and MIP-1α. (B) Potency of Ca²⁺ flux. Fura-2AM-loaded CCR1-HOS cells were stimulated with the indicated concentrations (0.1 to 50 nmol/L) of chemokines, and relative fluorescence was measured. The peak amplitude of Ca²⁺ response versus chemokine concentration was plotted in a semilog scale.