Apoptosis analysis using flow cytometry

An Annexin V-FITC apoptosis kit (BD Biosciences, NJ, USA) was used to determine the number of apoptotic cells according to the manufacturer’s instructions. Different cell groups were harvested with 0.25% trypsin and washed with PBS. After centrifugation, the cells were re-suspended in 100 μl buffer and then stained with Annexin V (3 μl) and propidium iodide (PI; 5 μl), and the mixture was incubated in the dark at 4 °C for 15 min. The cells were sorted using a FACS Calibur flow cytometer (BD Biosciences, USA), during the assay 10,000 cells per sample were collect and count. The result were analyzed using Cell Quest software (BD Biosciences, USA). The experiments were repeated three times.