Kinetics of the mesothelin-MUC16 interaction. (A) Binding of specified amounts of meso-Fc to OVCAR-3 was detected by flow cytometry. The left panel shows data for one representative experiment. Composite analysis of three independent experiments is shown in the panel on the right. Middle panel shows native expression of MUC16 on the OVCAR-3 cells used in this assay. (B) Time kinetics of meso-Fc (25 nM) binding to OVCAR-3 cells was determined by flow cytometry. After incubation with meso-Fc for the designated time intervals, the cell suspensions were diluted with 2 ml of buffer (a step that takes approximately 10 s) and analyzed. Raw flow cytometry data from one experiment is shown in panel on the left and composite analysis of two independent experiments is in the right panel. The middle panel shows expression of MUC16 on the OVCAR-3 cells used in this experiment. (C) Inhibition of meso-Fc binding to OVCAR-3 cells by soluble MUC16 was also measured by using a flow cytometry assay. Meso-Fc preincubated with designated amounts of MUC16 was added to the cells. Binding was analyzed by flow cytometry. Left panel shows raw data for one experiment and panel on right shows composite data of two independent assays. The middle panels show VK-8 binding to the OVCAR-3 cells used in this experiment in the presence (green) or absence (blue) of soluble MUC16 (100 nM).