Quantitative analysis of rel A DNA binding in CLL B cells and its relationship to apoptosis. (A) Subunit-specific Rel A ELISA (Active Motif) assays were performed using 2 μg of nuclear extracts derived from 30 freshly isolated ex vivo CLL samples. Absorbance was measured at 450 nm. Results were compared with a standard curve generated using recombinant protein that allowed the calculation of the amount of p65 present in CLL cells expressed relative to total nuclear protein from CLL patient samples. (B) Parallel experiments were performed in which aliquots of CLL B cells derived from the same 30 CLL patients were set up in in vitro culture for 48 hours, and spontaneous apoptosis was then measured using annexin V and PI staining. FACS plots were analyzed using Summit 4.3 software (Dako). Percentage apoptosis was defined as the total annexin V⁺ cells (PI⁺ and PI⁻) after 48 hours in culture. (C) The level of apoptosis showed a significant negative correlation with nuclear Rel A DNA binding activity in the nucleus of freshly isolated CLL cells.