Protocols for Apoptosis

Annexin V Assay: Apoptosis is characterized by a variety of morphological features. Changes in the plasma membrane are one of the earliest of these features. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the PM, thereby exposing PS to the external cellular environment. Annexin V is a 35-36 kD Ca2+ dependent, phospholipd-binding protein that has a high affinity for PS, and binds to cells with exposed PS. Annexin V may be conjugated to fluorochromes such as FITC so that flow cytometric anlysis of cells undergoing apoptosis can be performed.

Annexin V staining precedes the loss of membrane integrity which accompanies the latest stages of cell death resulting from either apoptotic or necrotic processes. Therefore, staining with Annexin V in conjunction with vital dyes such as propidium iodide (PI) or 7-amino-actinomycin D (7-AAD) allows the investigator to identify early apoptotic cells (Annexin V positive, PI or 7-AAD negative). However, the assay does not distinguish between cells that have already undergone an apoptotic death and those that have died as a result of a necrotic pathway because in either case the dead cells will stain with both Annexin V (+) and PI (+).

See sample Annexin V protocol   

Propidium Iodide (PI) is used to assess plasma membrane integrity in Annexin V apoptosis assays. PI is a fluorescent vital dye that stains DNA. It does not cross the PM of cells that are viable or in the early stages of apoptosis because they maintain PM integrity. In contrast, cells taht are in the late stages of apoptosis or already dead have lost PM integrity and are permeable to PI.