Cell cycle analysis using flow cytometry

MCF-7 human breast cancer cells were seeded at a density of 2 × 10⁵ cells/ml and then incubated overnight as described previously [15]. The medium was substituted by fresh DMEM containing several concentrations (0, 10, 50 and 100 µM) of glycyrrhizinic acid and then further incubated for 48 h. The treated and untreated cells were trypsinized, washed with cold PBS twice and then fixed using 70% methanol for 30 min. The cells were again washed with ice-cold PBS, and then stained with 20 µg/ml propidium iodide and then 10 µg/ml RNase A was added for 30 min. Finally, the cells were analysed using a FACSCalibur flow cytometer (FACSCalibur; BD Biosciences), and the data were processed by cell cycle analysis software (Modifit 2.0).