Flow-Cytometric Analysis of Cell-Cycle Distribution

For each dose-rate exposure, 1 × 10⁶ cells were harvested at 0, 2, 4, 6, 8, 10, 12 h after the start of irradiation and fixed with 70% ethanol, and then kept at 4 °C for at least 2 h. After a centrifugation, the cells were re-suspended in 1 ml phosphate-buffered saline (PBS) (−). After a centrifugation again, the DNA was stained with 0.5 ml FxCycle^TM propidium iodide (PI)/RNase staining solution (Life Technologies) including 0.2% v/v triton X for 15 min in the dark at room temperature. Cell-cycle distribution was then obtained by using the Attune acoustic focusing flow cytometer (Applied Biosystems by Life Technologies TM).

The fluorescence intensity emitted from the DNA in a nucleus was normalized by that from the DNA contained in a cell in G₀/G₁ phase. The cell-cycle distribution (fractions of the cells in G₀/G₁, S and G₂/M) was then obtained from the DNA profile. All sets of cell-cycle study were performed three times. By using the Tukey-Kramer method³², we evaluated if there is significant difference in the change of cell-cycle distribution from the control group (before irradiation at t = 0 (h)).