Monoclonal antibody (mAb)–induced cell death detected by flow cytometry is not an artefact of cell aggregation. Alternative assays used to detect mAb-induced cell death. (A) RAJI cells were treated with mAb (10 μg/mL) for 4 hours and labeled in situ with SYTOX green. Cell death was then analyzed by fluorescence microscopy (original magnification: ×4), representative of 2 independent experiments. (B) Cells were treated with mAb (10 μg/mL) for 24 hours and cell viability determined by the colorimetric XTT assay. Cells were incubated for 4 hours at 37°C with XTT reagent, and absorbance was normalized to untreated cells. Mean ± SEM of 3 independent experiments are shown. (C) Cells were pretreated with Cathepsin inhibitor III (100μM) for 30 minutes before treatment with mAb and after 4 hours cell death and cell aggregation were analyzed by flow cytometry (top panel) and microscopy (bottom panel), respectively (original magnification: ×4). Mean ± SEM of 3 independent experiments are shown. Cathepsin inhibitor III potently attenuated cell death induced by both tositumomab (anti-CD20) and L243 (anti-HLA DR) but had no effect on cell aggregation. NT indicates nontreated; Tos, Tositumomab/B1; and control mAb, anti-CD3 (OKT3).