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).