2.2 Apoptosis Assay by Fluorescence Activated Cell...
Apoptosis was determined quantitatively by flow cytometry using the Annexin Vfluorescein isothiocyanate(FITC)/propidium iodide(PI)kit(KeyGEN Biotechnology，Nanjing，China)according to the manufacturer's instruction.After 48h treatment，culture supernatants were collected for floating dying and apoptosis cells.Adherent cells were rinsed with phosphate buffered saline(PBS)and harvested by brief trypsinization.Cell fractions were combined，washed with PBS，and then doublestained with FITC-conjugated Annexin V and PI for cellular staining for 15min at room temperature.Flow cytometric analyses were performed on a FACS Calibur TM(Becton Dickinson，U.S.A.)within 1h.The apoptotic cells were measured as late or early apoptotic cells，which were shown respectively in the upper right or lower right quadrant of the FACS histogram.In the early stages of apoptosis，the cells were stained by Annexin V-FITC，but excluded by PI(FITC+/PI—)，because the cell membrane remained intact.While in the late stages，plasma membrane integrity is broken，and it enables PI to exist and bind to DNA and makes cells positive for both Annexin V-FITC and PI(FITC+/PI+).(https://www.daowen.com)