4.8. Analysis of Cell Cycle with Propidium Iodide Staining by Flow Cytometry

Cells (5 × 10⁵) of the different tumor cell lines were seeded in 6-well plates in a volume of 2 mL, and the following day exposed to the resveratrol isomers and SAHA at the above-mentioned concentrations. As control for apoptosis, STS stock solution (Enzo Life Sciences) was added 20 h before flow cytometry at a final concentration of 5 µM. After 72 h of incubation, cell cycle analysis was determined by PI staining. For this purpose, cells were collected after detachment with trypsin, together with medium and PBS used to wash the plates. Samples were centrifuged at 200 g for 4 min, the supernatant discarded, and the cell pellet washed once with cold PBS (4 °C). Subsequently, the cells were fixated with 1 mL ice cold 75% ethanol and mixed gently, and then placed in the fridge for at least 2 h. Thereafter, 2 mL of cold PBS was added and samples were centrifuged again (200 g, 10 min, 4 °C), followed by carefully aspirating the supernatant. Cells were then washed once with cold PBS and stained with PI/RNAse solution while gently vortexing (PI 50 µg/mL (Sigma–Aldrich) + 100 µg/mL RNAse (Sigma–Aldrich)), and then incubated for at least 20 min at 4 °C in the dark. A NovoCyte 2060R flow cytometer (Agilent Technologies Inc.) was used for measurements; data were analyzed with NovoExpress 1.4.1 software (Agilent Technologies Inc.). For each measurement, 1 × 10⁴ events were counted.