2.3 Detection of cell apoptosis by fluorescence ac...
Flow cytometric analysis was performed to detect the cell apoptosis of each group by using Annexin V-fluorescein isothiocyanate(FITC)/propidiumlodide(PI)kit(Qihaifutai Biotechnology，Shanghai，China)as recommended by manufacturer.After 48h treatment，culture supernatants which contained floating dying and apoptotic cells were collected，and then merged with the adherent cells which were rinsed with phosphate buffered saline(PBS)and harvested by a brief trypsinization.The mixture was washed with PBS，centrifuged for 5min at 2000 RPM，and the supernatants were discarded.Cells were re-suspended in 0.5mL binding buffer，and then double-stained with FITC-conjugated Annexin-V and PI for 15min at room temperature.Flow cytometric analyses were performed on a FACS CytomicsTM(FC500，Beckman Coulter，USA)within 1h.The apoptotic cells measured as late or early apoptotic cells were shown respectively in the upper right or lower right quadrant of the FACS histogram.(https://www.daowen.com)