BL22 cytotoxicity involves DNA-strand breaks, PARP cleavage, and damage to mitochondria. CLL cells were cultured for 72 hours in the presence of BL22 or LMB-9 at 1000 ng/mL or in the absence of immunotoxin. After staining with fluorescent compounds, cells were analyzed by flow cytometry. (A) Cells were stained for Annexin V, as described in the legend to Figure 2. (B) For analysis of the mitochondrial membrane potential Δψ_m, cells were incubated with DiOC6(3) for 30 minutes. Results from 1 representative experiment are displayed; 2 additional experiments gave similar results. (C) DNA-strand breaks were assessed using TUNEL assay, as described in “Patients, materials, and methods.” (D) CLL cells were grown for indicated periods in the presence of BL22 or in medium without immunotoxin. Cell lysates containing 50 μg protein were loaded into each lane, and cleavage of PARP was analyzed by immunoblotting, as described in “Patients, materials, and methods.” An additional experiment gave comparable results.