Effect of quercetin on bortezomib-induced oxidative and apoptotic events. Fresh primary CLL cells (10⁶/mL) were pretreated with 20 μM quercetin for 1 hour and then incubated with 20 nM bortezmib for 12 hours. Cells were washed twice with HBSS. For the measurement of ROS (A), cells were stained with both 40 μM HE (O₂^·̄) and 5 μM H₂DCF-DA (H₂O₂) at 37°C for 30 minutes. ROS production was measured by flow cytometry in the FL1-H and FL3-H channels. For the determination of ΔΨm (B), cells were incubated with 20 nM TMRM at 37°C for 15 minutes, and ΔΨm was measured in the FL3-H channel. For the annexin V assay (C), cells were resuspended in the binding buffer and then stained with annexin V–FITC and PI at room temperature for 15 minutes. Apoptotic cells were analyzed in both the FL1-H and FL3-H channels. Annexin V⁺/PI⁻ and annexin V⁺/PI⁺ cells were counted as apoptotic. The number in each chart indicates the percentage of cells that underwent apoptosis. (D-F) Statistical data from 5 CLL patients (D), the HRC57 cell line (E), and the DoHH2 cell line (F). Statistical significance of the inhibition of quercetin on bortezomib was analyzed by the t test. *P < .001.