Flow Cytometric Analysis of Cell Cycle

For flow cytometry, 10⁴ cells were treated with increased concentrations (34 and 68 µM for the T98 cell line and 30 and 60 µM for the U87 cell line) of 9″-lithospermic acid methyl ester. As a negative control, untreated cells containing less than 1% DMSO were employed. All samples were run in triplicate, and at least three separate experiments were conducted. Flow cytometric analysis was performed 72 h post-treatment with 9″-lithospermic acid methyl ester. For the DNA cell cycle, cells were treated with trypsin, centrifuged, washed with PBS twice, and then incubated with PI (Propidium Iodide) working solution (50 µg/mL PI, 20 mg/mL RNase A, and 0.1% Triton X-100, Sigma-Aldrich, St. Louis, MO, USA) for 15 min at 37 °C in the dark. With the use of a flow cytometer (Omnicyt Flow Cytometer, Cytognos, Athens, Greece), the PI fluorescence of 10⁴ individual nuclei was determined. Subsequently, the fractions of cells in G0/G1, S, G2/M, and sub-G0/G1 phases were analyzed [18 (link)].