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).