4.8. Determination of Cell Cycle by Flow Cytometry

Cell cycle was evaluated using propidium iodide (PI). Then, 48 h after transfection or 24 h after serum deprivation, 2 × 10⁶ cells were fixed with EtOH 70% at −20 °C for 12–24 h. Next, EtOH was removed and washed twice with fresh ice-cold PBS and the cells were permeabilized with PBS containing 0.1% (v/v) Triton X-100 and 100 µg/mL RNAasa at 37 °C for 15–30 min. The percentages of cell cycle stages were defined by incubating propidium iodide (2 µM) at 37 °C for 30 min in darkness, as per the manufacturer’s instructions. To analyze cell cycle status, cellular DNA content was estimated using a FACS-SCAN flow cytometer (Becton Dickinson, Madrid, Spain). Forward and side scattering were considered to select appropriated cells. The fluorescence emitted from cells was acquired at a wavelength of 555/624 nm (Ex/Em) for PI.4.9.

Analysis of statistical significance was performed using the Kruskal–Wallis test combined with Dunn’s post-hoc test (GraphPad Prism Windows 8, San Diego, CA, USA). For comparison between two groups, the Mann–Whitney U test (or Student’s t-test for the analysis of Ca²⁺ determinations) was used. A p-value < 0.05 was considered to be statistically significant.