2.4. Cell Cycle and Apoptosis Analysis by Flow Cytometry

MCF-7 cells were collected 24, 48 and 72 h after treatment. For cell cycle analysis, cells were fixed with 70% ethanol, treated with 1% RNase in TE buffer and finally stained with a hypotonic propidium iodide (PI) solution (50 µg/mL in PBS). Cell cycle analysis was performed using a flow cytometer (LSRFortessa, BD Bioscience, San Jose, CA, USA). Cell cycle distribution (percentage of cells) in cell debris (sub-G1) and G1, S, and G2/M phases of the cell cycle was analyzed using FlowJo software version 7.6 (Treestar, Ashland, OR, USA). To discriminate between apoptosis and necrosis, cells were detached with Trypsin/EDTA and stained with FITC Annexin V (BioLegend, San Diego, CA, USA) and PI (50 µg/mL). Then, necrotic and apoptotic cells were determined using a flow cytometer (LSRFortessa). Data were analyzed using FlowJo software version 7.6 (Treestar, Ashland, OR).