Flow cytometric analysis of surface-expressed GPIIb-IIIaCys655Tyr on CHO cells. (A) Expression of GPIIb-IIIaCys655Tyr on the surface of transfected CHO cells as measured by monoclonal antibodies directed against GPIIb (Tab) or the intact complex (AP2). Cells were incubated with the indicated antibody or normal mouse IgG (IgG) at a concentration 20 μg/mL at room temperature for 60 minutes. After washing, the cells were then incubated with fluorescein isothiocyanate (FITC)-conjugated F(ab′)2 fragments of goat-antimouse IgG for 30 minutes and analyzed by flow cytometry. (B) Binding of the LIBS antibody D3 to GPIIb-IIIa transfected CHO cells. Transfected CHO cells were incubated with LIBS antibody, D3 (20 μg/mL), in the presence of different concentrations of RGDW peptides. After washing, the surface-bound antibody was analyzed by the addition of FITC-labeled goat antimouse IgG, followed by flow cytometry. Note that functional expression of the D3 epitopes on these transfected CHO cells is in an RGD dose-dependent manner. Similar results were obtained from wild-type GPIIb-IIIa transfected CHO cells (not shown).
