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4) were incubated in cell culture medium containing Fluo-3, AM fluorescent dye (5 μg/mL, Molecular Probes), and probenicid (710 μg/mL, Sigma) for 1 h at 37 °C. Cells were washed with Hanks buffer containing HEPES (20 mM), BSA (0.1%), and probenicid, and then were resuspended in 90 μL of the same buffer. A ligand addition plate was prepared by adding 135 μL of chemokines at various concentrations for the titration. To test inhibition of calcium release by TAK-779, 2 μL of this antagonist at various concentrations was added to the cells upon resuspension and 20 nM hMCP-1 was used to activate CCR2B receptors. Both plates were placed into the Fluorescence Imaging Plate Reader (FLIPR, Coherent, Inc.). Chemokines were dispensed from addition plate into the cell plate and calcium release was measured by the Argon Laser at an excitation wavelength of 488 nm and an emission wavelength of 530 nm.
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