Cell cycle analysis by flow cytometry
This method distributes the cells in the sub-G1, G0/G1, S, and G2/M phases based on the amount of DNA in the cell. Increasing sub-G1 cell population of treated cells predicate to the enhancement of apoptotic cells15.
The KG1a cells (1×105 cells/ well) were seeded in 96 well plates and treated with
100 ?M concentrations of 4 -HBTC for 24, 48 and 72 h. At the end time, the K562 treated cells were washed twice with PBS, suspended in 70% ethanol at -20 °C. Finally, the percentage of cells in each phase was measured.
Flow cytometric assessment of apoptosis in KG1a cells
To confirm the outcomes of former analysis, apoptotic cells were quantified with FITC-AnnexinV and PI double staining method with analyzing of the presence of phosphatidyl serine on the outer surface of the apoptotic cell membrane14. The KG1a cells were seeded in 96-well cell culture plates for 24 hr prior to treatment and treated with 4 -HBTC for 24, 48 and 72 hr. Briefly, after double washing of treated and control cells with PBS, 12 × 105 cells were resuspended in 100 µl binding buffer (1x) and then 5 ?L of FITC-conjugated Annexin V and 10 ?L PI were added. Afterward, it was incubated for 15 min in the dark at room temperature and finally, analyzed by flow cytometry (Partec PAS)16.
Assay of intracellular ROS level
2′-7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) is one of the most widely used methods for measuring intracellular ROS generation This dye is a stable compound that readily diffuses into cells and hydrolyzed by intracellular esterase to DCFH which is trapped within cells. In the presence of H2O2 or low-molecular weight hydroperoxides, peroxidases can oxidize DCFH2 to 2′-7′-dichlorofluorescein (DCF). The level of peroxide produced can be estimated by an increase in DCF fluorescence at 530 nm when the sample is excited at 485 nm17, 18. In the present study, cells were treated with 100 ?M of the 4 -HBTC for 24- 72 hours. The cells were incubated with 50 ?l DCFH-DA (10 ?M) for 30 min. Then, after twice washing with PBS amount of DCFH-DA fluorescence was detected using flow cytometry