3.4.4. Cell Cycle Analysis

The cell cycle was analyzed using PI-stained DNA. Cells from the compounds 7 treated cultures were collected at 24, 48, and 72 h, trypsinized, washed in ice-cold PBS, fixed by the addition of 70% ethanol and left for 2 h at −20 °C. Thereafter, the cells were washed twice in PBS, stained with 50 μg/mL of propidium iodide (Sigma, Merck KGaA, Darmstadt, Germany) in PBS, treated with 10 µg/mL of RNAse and analyzed by flow cytometry using FACScan device (BD, USA). A total of 2000 events were collected. The results were analyzed using FlowJo 10 software (BD, Franklin Lakes, NJ, USA).

Apoptosis analysis

Apoptosis was analyzed by flow cytometry using a FACScan device (BD, USA). Cells were incubated with the compounds 7 as above, trypsinized, washed in ice-cold PBS, stained with annexin V-FITC (BD, USA, Franklin Lakes, NJ) and propidium iodide (Sigma, Merck KGaA, Darmstadt, Germany), incubated for 15 min, and analyzed.