Pi double staining kit

Cell cycle analysis was performed by PI-based measurements of cell DNA content using flow cytometry. Cells were treated with various concentrations of EE (0.001, 0.01, 0.02, 0.1 mg/ ml) and EO (0.005, 0.01, 0.02, 0.03, 0.04 mg/ml) (dissolved in DMSO) for /48/ h, followed by collection of both attached and detached cells. The pellet was rinsed twice with cold PBS and cells were fixed in 70% ice-cold ethanol overnight at 20°C. Fixed cells were then washed twice with PBS, and DNA was stained with PI(Sigma-Aldrich) staining solution (20µl of cell suspension were added to 2ml of staining solution) and incubated in the dark for 5 min. Flow cytometry analysis was carried out using BD FACSCalibur Flow Cytometer.
Annexin V/PI apoptosis assay. Cells were cultured (1x106 cells/ml) overnight in 25 cm2 cell culture flasks. Then, cells were treated with various concentrations of EE (0.001, 0.01, 0.02, 0.1 mg/ml) and EO (0.005, 0.01, 0.02, 0.03, 0.04 mg/ml) (dissolved in DMSO) for /48/ h. After treatment, both adherent and detached cells were collected and rinsed twice with cold PBS. The cell pellet was resuspended in 1 ml of annexin-binding buffer and incubated with 5 µl of Annexin V-FITC and 5 µl of PI for 15 min. The cells were analyzed by flow cytometry and data were analyzed through Cell Quest program. Annexin V-FITC and PI double staining kit were purchased from BD (USA).