4.7. Cell Cycle Analysis by Flow Cytometry

The cell cycle was determined by the quantitation of DNA content using the nucleic acid stain propidium iodide followed by flow cytometry analysis. Propidium iodide (PI) is a fluorescence dye that binds both types of nucleic acids, DNA and RNA, proportionally to the amount of material present in the nucleus. Cells were seeded at a density of 3 × 105 in six-well plates, as described above. Following overnight preincubation, cells were treated with ME and exposed to various temperature conditions for 24 h. After treatment, the culture media was removed, and cells were collected as described above. Cell cycle analysis was carried out using a CellCycleFlowEx^® Kit (Exbio, Vestec, Czech Republic) according to the manufacturer’s instructions. Cells were stained for 30 min with PI, and RNA was digested using RNAse. Next, flow cytometry analysis was conducted within 4 h using a BriCyte E6 flow cytometer (Mindray, Shenzhen, China). The cell cycle distribution was presented as the percentage of cells containing 2n (G1 phase), 4n (G2 and M phase), and between 2n and 4n (S phase), as determined by PI staining.