| Assay Method Information | |
| | Scintillation Proximity Assay |
| Description: | The CCR3 receptor binding assay was performed in a Scintillation Proximity Assay (SPA) design with the radioligand recombinant human 125lodine-eotaxin-1. Cell membranes of hCCR3 C1 cells were again homogenized by passing through a single use needle (Terumo, 23Gx1 ") and diluted in SPA incubation buffer in suitable concentrations (0.5 -10 μg protein/well) in 96 well microtiter plates (1450-514, Perkin Elmer). The SPA assay was set up in the SPA incubation buffer with a final volume of 200μl and final concentration of 25mM HEPES, 25mM MgCI2 6xH2O, 1 mM CaCI2 2xH2O and 0,1% bovine serum albumin . The SPA assay mixture contained 60 μl of the membrane suspension, 80 μl of Wheat Germ Agglutinin coated PVT beads (organic scintillator, GE Healthcare, RPNQ-0001 ) 0,2 mg/well) , 40 μl of recombinant human 125Jodine-eotaxin-1 (Biotrend), diluted in SPA buffer to a final concentration of 30.000 dpm per well, and 20 μl of the test compound (dissolved in DMSO dilutions). The SPA assay mixture was incubated for 2 h at room temperature. Bound radioactivity was determined with a scintillation counter (Micro Beta "Trilux", Wallac). Included were controls for total binding (no displacer added, Bo) and non-specific binding (NSB) by adding unlabelled recombinant human Eotaxin-1 (Biotrend, Cat #300-21 ) or a reference compound. Determination of the affinity of a test compound was calculated by subtraction of the nonspecific binding (NSB) from the total binding (Bo) or the binding in the presence of the test compound (B) at a given compound concentration. The NSB value was set to 100% inhibition. |
| Affinity data for this assay | |
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