Comparison of the ability of CD20 mAbs to redistribute antigens on the cell surface. (A-B) Translocation of CD20 into the Tx100-insoluble fraction of the cell. Daudi (A) or DOHH (B) cells were incubated with FITC-labeled CD20 mAb (10 μg/mL) for 15 minutes at 37°C and washed. After chilling on ice, half of each sample was treated with 0.5% Tx100 for 15 minutes on ice. Both samples (lysed, dark gray bars; and not lysed, light gray bars) were washed and analyzed by flow cytometry (mean fluorescence intensity [MFI]) to assess bound FITC-CD20 mAbs. (C) FRET determination: Daudi cells were labeled with an equimolar mixture of Cy3 (donor)- and Cy5 (acceptor)-conjugated CD20 mAbs at 37°C with or without C8-deficient (C8-def) serum. FRET, which measures the proximity of neighboring mAb molecules, was assessed by flow cytometric analysis. These graphs are representative of at least 3 experiments, each showing similar results.