Flow cytometry
The ratio of apoptotic cells was measured with an annexin
V–FITC/propidium iodide (PI) Detection Kit (KGA108; Key Gen Biotech,
Nanjing, China) according to the manufacturer’s instructions. Cells were
harvested and washed twice with PBS and then resuspended in 500 μl
binding buffer. After adding 5 μl annexin V–FITC and 2μl PI into the
cell suspension, respectively, at least 20,000 live cells were analyzed
on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).
Data were analyzed by using FlowJo software (FlowJo, Ashland, OR, USA).