Hu1D10 treatment of CLL cells leads to increased intracellular ROS formation, which is decreased by preincubation with antioxidants, cytochalasin B, or methyl-β-cyclodextran. (A) CLL cells were treated as follows: (i) media only, (ii) Hu1D10 plus antihuman Fc antibody (both 10 μg/mL) for 4 hours, (iii) pretreatment with 25 mM N-acetyl cysteine followed by Hu1D10 plus antihuman Fc antibody (both 10 μg/mL) for 4 hours, or (iv) pretreatment with 10 mM tiron followed by Hu1D10 plus antihuman Fc antibody (both 10 μg/mL) for 4 hours. Following treatment, cells were incubated for 30 minutes at 37°C with 5 μM dihydroethidium and then analyzed by flow cytometry. These data represent one of 4 CLL samples tested. (B) CLL cells were treated as follows: (i) media only, (ii) Hu1D10 plus antihuman Fc antibody (both 10 μg/mL) for 4 hours, (iii) pretreatment with cytochalasin B (20 μM) followed by Hu1D10 plus antihuman Fc antibody (both 10 μg/mL) for 4 hours. Following treatment, cells were stained with dihydroethidium and then analyzed by flow cytometry. These data represent one of 3 CLL samples tested. (C) CLL cells were treated as follows: (i) media only, (ii) Hu1D10 plus antihuman Fc antibody (both 10 μg/mL) for 4 hours, (iii) pretreatment with methyl-β-cyclodextran followed by Hu1D10 plus antihuman Fc antibody (both 10 μg/mL) for 4 hours. Following treatment, cells were analyzed for ROS as noted. These data represent one of 3 CLL samples tested.