2.5. Cell Cycle Analysis

For studying cell cycle analysis, U87-MG, U373-MG, and U138-MG cells were seeded at concentration of 20,000 cells/cm² in multiwell 6 plates and treated with CisPt and Pt-8AQ at 10 µM. After 24, 48, and 96 h (37 °C, 5% CO₂), DNA content for cell cycle phase detection was performed by comparing untreated cells and 24 h treated cells. Cells were suspended in phosphate-buffered saline (PBS) and fixed with 70% (v/v) ethanol for 1 h at 4 °C. After a further PBS wash, cells were suspended in propidium iodide 50 µg/mL in PBS. Cells were incubated overnight at 4 °C and analyzed by flow cytometry (Navios EX, Beckman Coulter, Brea, CA, USA). At least 10⁴ events per sample were acquired and analyzed using a specific software (Navios EX Softwar, Beckman Coulter). Data are expressed as the mean fluorescence intensity (MFI) of the specific antibody.