2.8. Cell cycle analysis by flow cytometry

PC3 cells (5000cells/cm²) were treated with different concentrations of NCX4040 (10μM–50μM) in the presence or absence of catalase and DETDC in T-25 flasks for 48 hrs. The cells were trypsinized and spun down at 1500 g for 5 min. Then, the cells were washed with PBS and centrifuged at 1500 g for 5 min. 0.5 ml of 70%ethanol was added to the cells which were kept at 4°C for overnight. The cells were centrifuged at 1500g for 5 min and were washed with PBS. The cells were treated with RNaseA and incubated at 37°C water bath for 30 min. The cells were washed with PBS and centrifuged for 5 min. The cell pellets were suspended in 100 μl PBS containing 100μg/ml PI (1 mg/ml) (Sigma). The cells were analyzed using a flow cytometer (FACS Calibur; Becton Dickinson, Mountain View, CA).