( brResults K d determination ...)The development of a CXCR2 chemokine receptorbinding assay using the LEADseeker Multimodality Imaging System

br> Results
K_d determination
Saturation binding was performed with dilutions of [¹²⁵I]GRO-αto give a range of concentrations from 2.5 to 2000 pM in the wells. Figure 1 shows the saturation curve that was fitted using non-linear regression with the data analysis software package GraphPad Prism v4.0. A K_d value of 383 pM (95% confidence intervals 318 to 447 pM) was estimated directly from the curve and B_max was determined at 6 pmol/mg of protein.

DMSO tolerance
Dilutions of DMSO were prepared in assay buffer to give a range of concentrations from 2% (v/v) to 25% (v/v) in the wells. From the results shown in figure 2 it can be seen that the assay was tolerant to DMSO up to a final concentration of 12.5% (v/v)

IC₅₀ determination
Competitive binding of 0.37-nM [¹²⁵I]GRO-αwith unlabeled IL-8 and GRO-αwas assessed and the IC₅₀ values calculated as shown in Figure 3. DMSO was included at an assay concentration of 2.5% (v/v). Final concentrations in the well for unlabeled GRO-αwere 0.03 to 440 nM, and for unlabeled IL-8, 0.03 to 500 nM.

The IC₅₀ value for GRO-αwas determined to be 2 nM (95% confidence interval range 1.7 to 2.3 nM) and the K_i value was 1.02 nM (95% confidence intervals 0.87 to 1.19 nM). The IC₅₀ value for IL-8 was 11 nM (95% confidence interval range 10 to 12 nM) and the K_i was 5.68 nM (95% confidence intervals 5.14 to 6.27 nM).

Time course
A time course was performed using standard reagent concentrations as detailed in the protocol and DMSO included at an assay concentration of 2.5% (v/v). Readings were taken over an increased incubation time of 18 h. The assay reached equil
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