Uorescence activated cell sorting ow cytometry

To assess the cell cycle and apoptosis, 3×105 treated cells were seeded into 6-well plates and cultured for 48h at 37°C. The cells for cell cycle analysis were digested using trypsin (Hyclone), washed twice with phosphate-buffered saline (PBS), and xed in 70% ethanol overnight at 4°C. The cells were centrifuged at 500g for 5min, washed twice with cold PBS, and centrifuged. After treating with RNase A (0.1mg/ml) and propidium iodide (PI, 0.05mg/ml) purchased from 4A Biotech (Beijing, China) for 30min at 37°C, cell cycle analysis was performed through uorescence-activated cell sorting ow cytometry (Beckman Coulter, Palo Alto, CA, USA). For the analysis of apoptosis, cells were trypsinised followed by two PBS washing steps. The cells were stained using the Annexin V/PI detection kit (4A Biotech, Beijing, China) for 5min at room temperature. The apoptotic cells were measured using ow cytometry (Beckman Coulter). All experiments were repeated at least three time.

To assess the cell cycle and apoptosis, 3 × 10 5 treated cells were seeded into 6-well plates and cultured for 48 h at 37°C. For cell cycle analysis, the cells were digested using trypsin (Hyclone, Logan, UT, USA), washed twice with phosphate-buffered saline (PBS), and xed in 70% ethanol overnight at 4°C. The cells were centrifuged at 500 ×g for 5 min, washed twice with cold PBS, and centrifuged. After treating the cells with RNase A (0.1 mg/mL) and propidium iodide (PI, 0.05 mg/mL, 4A Biotech, Beijing, China) ) for 30 min at 37°C, cell cycle analysis was performed using uorescence-activated cell sorting ow cytometry (Beckman Coulter, Palo Alto, CA, USA). To analyze apoptosis, the cells were trypsinized followed by two PBS washes. The cells were stained using the Annexin V/PI detection kit (4A Biotech) for 5 min at 25°C. Apoptotic cells were measured using ow cytometry (Beckman Coulter). All experiments were repeated at least three times.