Viability of FL5.12 clones expressing Flag-Bcl-2 or Flag-Bcl-XL after vinca alkaloid treatment. (A) Cells were seeded at 1 × 105 cells/mL in triplicate wells in media containing IL-3 and increasing concentrations of vincristine. Cells were lysed at 72 hours in a hypotonic buffer containing propidium iodide (PI) (0.01 mg/mL) and the percent of nuclei with fragmented DNA was determined by flow cytometric analysis. Viability (percent of cells with nonapoptotic nuclei) is shown as the mean ± SD of triplicate cultures. No statistical significance was found between Flag-Bcl-2 and Flag-Bcl-XL clones at each concentration of drug (P < .05, Student's t-test). The graph shown is representative of at least two separate experiments. (B) Cell viability was determined as above at each time point following continuous incubation in media containing IL-3 and 10 μg/mL of vincristine. Cells were seeded at 1 × 105 cells/mL in triplicate wells and lysed in a hypotonic buffer containing propidium iodide (0.01 mg/mL). Viability (percent of cells with nonapoptotic nuclei) is shown as the mean ± SD of triplicate cultures. No statistical significance was found between Flag-Bcl-2 and Flag-Bcl-XL clones at each time point (P < .05, Student's t-test). The graph shown is representative of at least two separate experiments. (C) Cells were seeded at 1 × 105 cells/mL in triplicate wells in media containing IL-3 and increasing concentrations of vinblastine. Cells were lysed at 72 hours in a hypotonic buffer containing propidium iodide (0.01 mg/mL) and the percent of nuclei with fragmented DNA was determined by flow cytometric analysis. Viability (percent of cells with nonapoptotic nuclei) is shown as the mean ± SD of triplicate cultures. No statistical significance was found between Flag-Bcl-2 and Flag-Bcl-XL clones at each concentration of drug (P < .05, Student's t-test). The graph shown is representative of at least two separate experiments. (D) Cell viability was determined as above at each time point following continuous incubation in media containing IL-3 and 10 μg/mL of vinblastine. Cells were seeded at 1 × 105 cells/mL in triplicate wells and lysed in a hypotonic buffer containing propidium iodide (0.01 mg/mL). Viability (percent of cells with nonapoptotic nuclei) is shown as the mean ± SD of triplicate cultures. No statistical significance was found between Flag-Bcl-2 and Flag-Bcl-XL clones at each time point (P < .05, Student's t-test). The graph shown is representative of at least two separate experiments. The percentage of apoptotic nuclei was determined at different time points in triplicate cultures by nuclear PI staining followed by flow cytometric analysis. In this assay, the nuclei of apoptotic cells exhibit a sub-G0 profile characteristic of DNA fragmentation. Percentage of nonapoptotic nuclei is expressed as viability. Analysis was performed using Lysis II software (Becton Dickinson, San Jose, CA) on a FACScan flow cytometer. Results were based on the analysis of at least 5 × 104 events from each triplicate culture.
