Analysis of Notch1, Notch2, and Hes1 expression in B-CLL cells cultured ex vivo. (A,B) Freshly isolated B-CLL cells were cultured for 24 hours in complete medium (n = 8), and apoptosis was determined by flow cytometric analysis of annexin V/PI–stained cells. Results in panel A (mean ± SD of all 8 patients) and panel B (relative to patient 6 and representative of 8 patients) are presented as the percentage of viable (annexin V⁻/PI⁻), early apoptotic (annexin V⁺/PI⁻), late apoptotic (annexin V⁺/PI⁺), and necrotic cells (annexin V⁻/PI⁺). (C) Cultured B-CLL cells were separated into viable and apoptotic fractions (n = 4) using annexin V–coated magnetic microbeads, which bind to cells with external exposure of membrane phosphatidylserine. Flow cytometric analysis of B-CLL cells stained with annexin V–FITC shows unfractioned cells (i), negatively selected viable cells (ii), and positively selected apoptotic cells (iii). The data shown for patient 6 are representative of 4 patients. (D) Whole-cell lysates (25 μg) extracted from the indicated populations were analyzed by Western blot for Notch1, Notch2, and Hes1 expression (n = 4), and protein loading was assessed by reprobing the blots with an anti–β-actin mAb. Densitometry was performed, and the density of the bands corresponding to Notch2-precursor, Notch2-TM, Notch2-IC, and Hes1, expressed as arbitrary units, is reported for each cell population. Densitometry units (U) were calculated relative to β-actin for each lane. The data shown for patient 6 are representative of 4 patients.