Cytotoxic activity of APO866 in primary leukemia cells, MOLT4, and U266 cells. (A) Primary B-CLL (> 80% CD19⁺ cells, n = 23), T-CLL (> 90% CD3, CD2, CD5, CD7⁺, n = 1), and AML (> 70% blasts, n = 5) cells were isolated from peripheral blood (PB) samples by density gradient centrifugation. 10⁶ cells/well were seeded in 24-well plates and cultured with or without 10 nM APO866 or 5 μg/mL fludarabine. Ninety-six hours later cells were harvested, washed, stained with fluorescein isothiocyanate (FITC)–conjugated annexin-V and PI, and analyzed by flow cytometry. (B) Quantification of early apoptotic (annexin-V⁺/PI⁻) and late apoptotic/necrotic (annexin-V⁺/PI⁺) cells on exposure to 10 nM APO866. The percentage of early apoptotic cells is shown as ▬ while late apoptotic cells are shown as ▭. (C) 3 × 10⁴ MOLT4 and U266 cells/well were plated in 96-well plates and incubated with or without APO866 at the indicated concentrations for 96 hours. Thereafter, cell death was quantified by MTT colorimetric assay, and by AV/PI staining and flow cytometry. Results are presented as means of triplicate wells and SD.