Apoptosis-Induced Changes in Plasma Membrane
The apoptotic process is characterized by certain morphological features. The
features include changes in the plasma membrane (such as loss of membrane symmetry and loss of membrane attachment), a condensation of the cytoplasm and nucleus, protein cleavage, and internucleosomal cleavage of DNA. In the final stages of the process, dying cells become fragmented into “apoptotic bodies” and consequently eliminated by phagocytic cells without significant inflammatory damage to surrounding cells.
Phosphatidylserine Binding Assays Using Annexin V Conjugates
and can be detected by flow cytometry and cell imaging through binding to fluorochrome-labeled Annexin V conjugates when calcium is present. Annexins are a family of calcium-dependent phospholipid-binding proteins. They are abundant in eukaryotic organisms belonging to a family of ubiquitous cytoplasmic proteins involved in signal transduction. Annexin V’s preferential binding partner is phosphatidylserine, which is usually kept on the inner-leaflet (the cytosolic side) of cell membranes. In apoptosis, phosphatidylserine is transferred to the outer leaflet of the plasma membrane. The appearance of phosphatidylserine on the cell surface is a universal indicator of the initial/intermediate stages of cell apoptosis and can be detected before morphological changes can be observed.
Annexin V preferentially binds to negatively charged phospholipids such as phosphatidylserine (PS). In live cells, PS is located on the inner leaflet of the plasma membrane. During early apoptosis, loss of phospholipid asymmetry leading to exposure of PS on the external leaflet of plasma membrane. During late apoptosis, plasma membrane integrity is lost, therefore cell impermeant DNA dyes such as Propidium Iodide (PI) and 7-Aminoactinomycin D (7-AAD) could enter the cell. Therefore, by staining cells with a combination of fluorescently labelled Annexin V and PI/7-AAD, it is possible to detect non-apoptotic live cells (annexin V negative/PI negative), early apoptotic cells (annexin V positive, PI negative) and late apoptotic or necrotic cells (PI positive) by flow cytometry.