Characterization of anti-CD20 mAb. (A) SU-DHL-4 cells at 1 × 106/mL were incubated with mAb at 0.4, 2, and 10 μg/mL at room temperature for 15 minutes. Normal human serum was added as a source of complement, cells incubated at 37°C for 30 minutes, then propidium iodide added, and cells analyzed for cell lysis by flow cytometry. Data are representative of 3 independent experiments. (B) Examples of homotypic adhesion produced by the different mAbs after incubation with Raji cells for 4 hours at 37°C. Cells in tissue culture plates were viewed with an Olympus CKX21 inverted microscope (Olympus, Watford, United Kingdom) using a 10×/0.25 PH lens. Images were acquired using a CCL2 digital cooled camera (Olympus) and were processed with Cell B (Olympus Soft imaging solutions) and Adobe Photoshop version CS2 software (Adobe Systems, San Jose, CA). Original magnification 20×. (C) Rit m2a or tositumomab (Tosit) was added to hCD20 Tg B cells for 30 minutes on ice, and then the proportion of mAb that had redistributed to the Triton X-100-insoluble fraction was determined by flow cytometry. Bars show the mean plus or minus SEM for triplicate samples. (D) hCD20 Tg splenocytes were labeled with CFSE, incubated with nothing (no mAb), and irrelevant mAb (WR17), Rit m2a, 1F5, or tositumomab (Tosit) and subjected to a 1-hour phagocytosis assay with thioglycollate-activated peritoneal macrophages. Bars represent the mean plus or minus SEM for triplicate samples.