Raji cells were preincubated with concanamycin A (CMA) (0.01–100 nM) (A) or bafilomycin A1 (0–100 nM) (B) for 30 minutes before being treated with tositumomab or L243 (10 μg/ml). Cell death (AnV/PI⁺ cells) was measured 24 (A) or 4 (B) hours after treatment. (C) Inhibition of lysosomal V-type ATPase protects cells from long-term cytotoxicity induced by anti-CD20 and HLA-DR mAbs. Cells were pretreated with lysosomal V-type ATPase inhibitor concanamycin A (0.1 nM) for 45 minutes, and then mAbs were added. Cell viability was assessed 24, 48, and 72 hours later using an XTT assay (Roche). The plot represents mean metabolic activity relative to nontreated control from 2 independent experiments in 3 technical replicates. Data are shown as mean + SEM. (D) Impact of vATPase inhibitors on the volume of the cellular lysosomal compartment. Cells were incubated with concanamycin A (1 nM) or bafilomycin A1 (50 nM) for 30 minutes, and then mAbs were added (10 μg/ml). Subsequently, cells were labeled with LysoTracker red for 1 hour as described in Methods. Volume of lysosomal compartment was measured by flow cytometry as the magnitude of fluorescence in the FL2 channel. Level of fluorescence of Raji cells not labeled with LysoTracker (red histograms) was used as the reference control. Histograms represent FL2 fluorescence values of LysoTracker-labeled control cells with (green) or without (black) the indicated vATPase inhibitors as well as Ab-treated cells with (purple) or without (blue) vATPase inhibitors.