2.6 Flow cytometric assay
For cell cycle analysis, pancreatic cancer cells were seeded in 6 cm dishes and incubated overnight. The cells were treated with indicated concentrations of TA for 24 h. The treated cells were collected and washed twice with cold PBS. Following that, the cells were fixed with pre-cold 70% ethanol at 4 °C overnight. The fixed cells were washed one time with PBS and then resuspended in PBS containing PI (50 μg/mL) and DNase-free RNase (100 μg/mL), and incubated at room temperature for 30 min in the dark. Subsequently, the cell cycle analysis was performed using the FACSCalibur flow cytometer (BD Biosciences, CA, USA). The percentage of cells in different cell cycle phases was determined by the ModFit LT software (Verity Software House, Topsham, ME, USA).
The apoptosis rate of tumor cells was detected by flow cytometry with Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, San Jose, CA), following the manufacturer’s instructions. Pancreatic cancer cells were cultured in six-well plates. After reaching 70-80% confluence, the cells were treated with indicated concentrations of TA for 48 h. After that, the cells were harvested, washed twice with cold PBS, and resuspended in 100 μL of 1 × binding buffer. The cells were stained with 5 μL of Annexin V-fluorescein isothiocyanate and 5 μL of PI at room temperature for 15 min in the dark. Finally, 400 μL of 1×binding buffer was added to the cell suspension. The apoptosis rate was detected using the FACSCalibur flow cytometer (BD Biosciences, CA, USA). The data were analyzed with the FlowJo software (TreeStar, Inc., Ashland, OR, USA).