Binding of transfected CHO cells to soluble and immobilized Fg. (A) Binding of transfected CHO cells to FITC-labeled Fg. Soluble Fg was labeled with FITC. Transfected CHO cells were incubated for 45 minutes at room temperature with FITC-labeled Fg at a concentration of 100 μg/mL in the presence or absence of D3 (200 μg/mL) followed by flow cytometry. The left panel shows the binding of FITC-Fg in the presence or absence of antibody D3. The right panel shows the surface expression of GPIIb-IIIa complexes as detected by AP2. (B) Cell adhesion to immobilized Fg ligand. Transfected CHO cells expressing different allelic forms of GPIIb-IIIa complex were labeled with calcein-AM at 37°C for 30 minutes. After washing, the cells were allowed to attach for 60 minutes to wells coated with various concentrations of Fg, BSA, or anti-GPIIb-IIIa complex antibody at 37°C. After adhesion, nonadherent cells were removed by washing, and adherent cells were measured in a fluorescence plate reader. Nonspecific cell adhesion on BSA-coated wells was subtracted. Cell adhesion was normalized to cell adhesion on AP2 (Fg/AP2 ratio). CHO cells expressing both allelic forms of GPIIb-IIIa bound to immobilized Fg in a dose-dependent manner. (C) Adhesion of transfected CHO cells to immobilized Fg is mediated by GPIIb-IIIa. Cells were pretreated with AP2 or anti-PECAM-1 antibodies (20 μg/mL) for 30 minutes at room temperature. After washing, cell adhesion to 2 μg/mL Fg was measured and plotted by fluorescence intensity. AP2 but not anti-PECAM-1 antibody blocked cell adhesion mediated by both allelic forms of GPIIb-IIIa to immobilized Fg.