DART molecules mediate the cell-cell association. Jurkat T cells expressing surface CD3 were loaded with CellTrace CFSE dye, and cell membranes of CD19⁺ Daudi B cells were labeled with the fluorescent dye PKH26. (A) Cells were incubated for 30 minutes at room temperature with or without DART or BiTE molecules at 0.01 μg/mL. The Jurkat-Daudi cell-cell association was determined using flow cytometry and quantified as the percentage of positive cells in the upper right quadrant of FL1 versus FL2, representing the CFSE⁺PKH26⁺ double-positive population. (B) The experiment was repeated using increasing concentrations of the molecules. Each experimental point was set up in duplicate and the mean ± SD was plotted.