Cell cycle progression and Fluorescence Activated Cell Sorting (FACS) analysis profiles of AAV2 infected breast cancer cell lines. Cells were synchronized by trypsinization of 80% confluent cultures and plating at a density of 1 × 106 cells in E medium. The cells were incubated for 10 to 12 h at which point at least two-thirds of the cells are maximally synchronized in the G1 phase. This time point was designated time zero (t = 0). (A) MCF-7 (B) MDA-MB-468 and (C) MDA-MB-231 cells were infected with AAV2 at this point, and further cultured over a 7-day period. Both control and AAV2 infected cells were passaged 1:2 on day 2 and day 5. On each day, cells were harvested by trypsinization, washed with PBS, fixed in 70% ethanol, and stored at -20°C for 24 h. For performing FACS analysis, cells were resuspended in PBS containing 0.1% Triton X-100, 200 μg/ml DNase-free RNase A, and 100 μg/ml of propidium iodide for 30 min at 37°C. Flow cytometric analysis of 106 cells was carried out in a fluorescence-activated cell sorter, and the percentages of cells in theG1, S and G2/M phases of the cell cycle were determined using the Cell Quest program of Becton Dickinson. Data were analyzed with the Mod Fit LT program. For each cell line, FACS analyses were repeated three times. Results shown represent averages determined from three individual experiments (-, no data), with standard deviations presented in parentheses.