Annexin V-FITC/PI Apoptosis Assay

Analysis of apoptosis percentage was performed using an Annexin V-FITC/PI apoptosis kit (KeyGen Biotech, China) as previously described protocol [19 (link)]. Briefly, the cells were incubated with the rat medicated control sera, the WYHZTL sera and XAV939 plus the WYHZTL sera respectively for 24, 48 and 72 h. The sera volume ratio is same as above. When reaching 80 % confluence, cells were trypsinized, washed with PBS and resuspended in 500 μl of Annexin V-binding buffer. A mixture of 5 μl of FITC-labeled Annexin V plus 5 μl of Propidium Iodide (PI) solution was then added into the cells to incubate for 15 min at room temperature in the dark prior to flow cytometry analysis. Cell apoptosis was analyzed using CellQuest software (Becton-Dickinson). Three separate experiments were performed for each clone.