4.7. Cell Cycle Arrest Analysis by Flow Cytometry

Cell cycle arrest evaluation was carried out using a Cell Cycle Arrest Detection Kit (Sigma-Aldrich, Becton Dickinson, Franklin Lakes, NJ, USA), according to the protocol provided. MCF-7 cells at a concentration of 1 × 10⁶ cells/mL were harvested and transferred into 17 × 100-mm tubes. The cells were centrifuged for 5 min at 300× g, at room temperature, and the supernatant was aspirated. Buffer solution (1 mL) was added and the cells were resuspended by gentle vortexing, at low speed. The concentration was adjusted to 1.0 × 10⁶ cells/mL, with buffer solution. The cell suspensions were centrifuged at 300× g for 5 min at room temperature, and the supernatant was decanted carefully. Solution A (containing trypsin buffer), at a volume of 250 µL, was added to each tube and gently mixed by hand tapping the tube. Solution A was allowed to react for 10 min at room temperature. Two hundred µl of Solution B (containing trypsin inhibitor and RNase buffer) was added into each tube and gently mixed by hand tapping, and a further 10 min incubation was carried out, at room temperature. Cold Solution C (containing propidium iodide stain solution, 200 µL) was added into each tube. The solution was mixed, as above, and incubated once again for 10 min, on ice, in the dark. The tubes were finally capped and stored at 2–8 °C, in the dark, until flow cytometric analysis was performed. The samples were tested within 3 h, following the addition of Solution C. After storage, the samples in the tubes were mixed by hand tapping, to resuspend the cells. The cells were then analyzed using the FACS Canto II Becton-Dickinson (Franklin Lakes, NJ, USA) flow cytometer, by analyzing at least 10,000 cells per sample. The percentage of cells in G1, S, and G2 phases were analyzed by the ModFit LT software (Verity Software House, Topsham, ME, USA).