How to distinguish apoptosis from necrosis?

Apoptosis is a form of programmed cell death that is used by the body to remove unwanted, damaged, or senescent cells from tissues. In normal cells, the negative phospholipids reside on the inner side of the cellular membrane while the outer surface of the membrane is occupied by uncharged phospholipids. After a cell has entered apoptosis, the negatively charged phospholipids (PS) are transported to the outer cell surface by a hypothetical protein known as scramblase. Phagocytic white blood cells express a receptor that can bind to and detect the negatively charged phospholipids on the apoptotic cell surfaces. After detection, the apoptotic cells are removed. Necrosis has been characterized as passive, accidental cell death resulting from environmental perturbations with uncontrolled release of inflammatory cellular contents.

Annexin Ⅴ is a Ca²⁺ dependent phospholipid binding protein with a molecular weight of 35-36 KD, which can specifically bind to PS on the outer cell suiface with high affinity during apoptosis. Labeling of Annexin Ⅴ with fluorescent or radioactive molecules makes it possible to detect binding of labeled Annexin Ⅴ to the cell surface of apoptotic cells. After binding to the phospholipid surface, Annexin Ⅴ assembles into a trimeric cluster. This trimer consists of three Annexin Ⅴ molecules that are bound to each other via non-covalent protein-protein interactions. The formation of Annexin Ⅴ trimers results in the formation of a two-dimensional crystal lattice on the phospholipid membrane. This clustering of Annexin Ⅴ on the membrane greatly increases the intensity of Annexin Ⅴ when labeled with a fluorescent or radioactive probe. Propidium iodide (or PI) is a fluorescent nucleus dye, impermeant to live cells and apoptotic cells, but stains dead cells with red fluorescence, binding tightly to the nucleic acids in the cell. In molecular biology,  is a test to quantify the number of cells undergoing apoptosis. The assay uses the protein Annexin Ⅴ to tag apoptotic and dead cells, and the numbers are then counted using either flow cytometry or a fluorescence microscope.

Figure 1. Annexin V-FITC and pyridine iodide staining of L-929 Cells

In molecular biology, combining AnnexinⅤlabeling with fluorescent with PI can distinguish apoptosis from necrosis: Live cells (AnnexinⅤand PI both negative); Apoptonic cells (AnnexinⅤpositive, PI negative); Necrotic cells (AnnexinⅤand PI both positive). The one-step staining procedure takes only 10--15 minutes. Detection can be analyzed by flow cytometry or by fluorescence microscopy (Figure 1).