(A) Detection of total lysosomal volume in cells treated with mAbs. Cells were incubated with anti-CD20 mAb tositumomab or anti–HLA-DR mAb L243 as described previously (10 μg/ml). After that, cells were labeled with LysoTracker red and the volume of the lysosomal compartment measured by flow cytometry and fluorescence microscopy after 1 hour or 4 hours. Insert: L243-treated cell displaying a single large lysosome. (B) Relationship between lysosomal volume and cell death. Cells were treated as in A and subsequently, after 4 hours, costained with annexin V–Cy5.5 and assessed by 2-channel flow cytometry. (C) Detection of lysosomal membrane permeabilization followed by permeabilization of cytoplasm. Cells were treated with mAbs as described above and then stained with acridine orange (AO). The relative increase or decrease in FL1 fluorescence was assessed by flow cytometry. (D) Permeabilization of plasma membrane as detected by destained cytoplasm (arrows) of tositumomab-treated cells. Cells were stained with AO as described and then assessed by fluorescence microscopy. Original magnification, ×40. (E) Confocal microscopy of cathepsin B staining (red) 4 hours after treatment with tositumomab or L243 mAbs. DNA was counterstained with DAPI (blue). Bottom right-hand panel shows L243-treated cells costained with cathepsin B (red) and LAMP-1 (green) with control-treated cells shown in the insert. A gross increase in the cathepsin B signal and peripheral localization of LAMP-1 (arrows) was observed. Scale bars: 20 μm. (F) The impact of cathepsin inhibitor III on cell death induced by tositumomab and L243. Cells were pretreated with different concentrations of inhibitor, and cell death (percentage of annexin V–FITC/PI–positive cells) was assessed 4 hours after treatment. Data represent the average of 3 independent experiments + SEM.