5.10. Cell Cycle Analysis by Flow Cytometry

U87MG cells were plated in DMEM 0.5% FBS (5 × 10⁵ cells/well) for 48h and treated with Iso 15 and 25 µM. At 24, 48, and 72 h of treatment, cells were washed in sample buffer (0.1% Glucose in HBSS without Ca⁺⁺ and Mg⁺⁺), resuspended in sample buffer and fixed in ice-cold 70% ethanol overnight at 4 °C. After centrifugation, cells were incubated in propidium iodide (PI) staining solution (50 µg/mL of PI) for 30 min at room temperature and analyzed within 24 h in a flow cytometer (Gallios Instrument, Beckman Coulter, Brea, CA, USA). Cell cycle analysis was performed by using Kaluza software for analysis v. 2.1 (Beckman Coulter).