Analysis of Apoptosis and Necroptosis by Fluorescence-Activated Cell Sorting

Fluorescence-activated cell sorting (FACS) is a laser-based, biophysical technology that allows simultaneous multiparametric analysis. For the analysis of dying cells, fluorescently labeled Annexin V (Annexin V(FITC)) and propidium iodide (PI) are the most commonly used reagents. Instead of PI, 4',6-diamidino-2-phenylindole (DAPI) can also be used. DAPI is a fluorescent stain that binds strongly to A-T-rich regions in DNA. DAPI and PI only inefficiently pass through an intact cell membrane and, therefore, preferentially stain dead cells. DAPI can be combined with Annexin V(FITC)and the potentiometric fluorescent dye, tetramethylrhodamine methyl ester (TMRM), which measures mitochondrial permeability transition and mitochondrial membrane depolarization. TMRM is a cell-permeable fluorescent dye that is sequestered to active mitochondria, and hence labels live cells. On apoptosis or necroptosis the TMRM signal is lost. The advantage of using Annexin V(FITC)/DAPI/TMRM is that the entire cell population is labeled, and it is easy to distinguish living (TMRM + /Annexin V(FITC)-/DAPI-) from dying or dead cells (apoptosis: TMRM-/Annexin V(FITC)+ /DAPI-; necrosis: TMRM-/Annexin V(FITC)+ /DAPI+). This is important because cell debris (fluorescent negative particles) must be avoided to establish the correct parameters for the FACS analysis, otherwise incorrect statistical values will be obtained. To obtain information on the cell concentration or absolute cell counts in a sample, it is recommended to add an internal microsphere counting standard to the flow cytrometric sample. This protocol describes the FACS analysis of cell death in HT1080 and L929 cells, but it can be readily adapted to other cell types of interest.