3.2.4. Analysis of Cell Cycle by Flow Cytometry

For these experiments, HeLa cells were used, which were seeded in 6-well plates at a concentration of 5 × 10⁵/well in a final volume of 2 mL culture medium. The cells were then treated with the test compounds for 24 h at the indicated concentrations. After this incubation period, the cells were detached with trypsin-EDTA and harvested by centrifugation. The pellet thus obtained was fixed in 70% ice-cold ethanol.

After this incubation period, the cells were detached with trypsin-EDTA and harvested by centrifugation. The pellet thus obtained was fixed in 70% ice-cold ethanol for at least 1 h. The cells thus fixed were treated with a 0.1% v/v solution of Triton_X-100 in phosphate buffered saline (PBS) containing RNAseA and propidium iodide (PI) at the final concentration of 0.02 mg/mL. The cells were incubated at room temperature for 30 min and then analyzed on a Cytomic FC500 flow cytometer (Beckman Coulter) in the FL3 channel. DNA histograms were analyzed using MultiCycle for Windows (Phoenix Flow Systems, San Diego, CA, USA).