Cell Cycle Analysis by Flow Cytometry

The analysis of the cell cycle was performed by FC using PI staining. Assays were prepared in 12 well plates by adding 500 μL (1 × 10⁶ cells/well) of complete medium and 500 μL of prepared eluates and then incubated for 24 and 48 h. Cells treated with 1 μM of the cell cycle arresting factor nocodazole (Sigma-Aldrich Corp., St. Louis, MO, USA) for 16 h constituted a positive control, whereas cells cultured in a complete medium for 24 h and 48 h constituted a negative control. Cells were washed twice with cold PBS (Sigma-Aldrich Corp., St. Louis, MO, USA) and then fixed with ice-cold 70% ethanol at −20 °C for 20 min. Afterwards, cells were treated with RNase A DNase & Protease-free (10 mg/mL) (Canvax Biotech, Córdoba, Spain) and incubated at 37 °C for 1 h before staining with PI solution (10 μg/mL) (Sigma-Aldrich Corp., St. Louis, MO, USA). After a 30 min incubation at 4 °C, the percentage of cells in each cell cycle phase were assessed using Kaluza analysis 1.5 A software (Beckman Coulter). On the DNA content histograms, the number of cells was plotted on the y-axis, whereas the DNA content, as measured by PI fluorescence, was depicted on the x-axis [35 (link)].