Figure 5

Binding of multimeric CD200 ligands to CD200R proteins. (A). Diagram of the reverse phase array depicting immobilized CD200R interacting with the multivalent bead ligand.(B). A representative microarray set showing binding of hCD200 beads to CD200R-CD4d3+4 proteins, but not control rat CD4.(C). Mean fluorescence intensity ± SEM of all four sets is shown versus receptor protein concentration arrayed. Serial two-fold dilutions of purified, soluble, recombinant human, mouse and rat CD200R-CD4d3+4 proteins, and of control rat CD4 were arrayed onto epoxy-coated microscope slides. Each receptor protein dilution series was arrayed in 3 rows of 4 spots, ranging in concentration from 40 μg ml^-1 (first spot) to 0.08 μg ml^-1 (spot 10), with control spotting buffer containing 0.5 mg ml^-1 BSA in the last two spots. Only the first 8 dilutions (2 rows) are shown in (B) and analyzed in (C). All arrays were performed in quadruplicate. All receptors were arrayed on the same slide, which was incubated for 16 h at 4°C with polyvalent human CD200-CD4d3+4 FITC-fluorescent beads. At the highest concentrations, the hCD200R spots appear white (saturating conditions). Quantitative measurements are expressed as fluorescence units at 532 nm (green) versus amount of protein arrayed.