Annexin V-PI Apoptosis Assay

Apoptosis-mediated cell death was examined by a double staining method, using a FITC-labeled Annexin V/propidium iodide (PI) apoptosis detection kit (BD Bioscience, San Jose, CA), as previously described [14 (link)]. Briefly, cells were treated for 12 h and then harvested, washed in cold phosphate-buffered saline (PBS) twice and then stained with fluoresceinisothiocyanate (FITC)-conjugated Annexin V and PI dyes. The externalization of phoshotidylserine and the permeability to PI were evaluated using a FACS Calibur flowcytometer (BD Bioscience). Data from 10,000 gated events per sample were collected. Cells in early stages of apoptosis were positively stained with Annexin V, whereas cells in late apoptosis were positively stained with both Annexin V and PI.