Annexin V is a protein that binds specifically to phosphatidylserine, a lipid molecule in cell membranes. This interaction is the basis of a scientific method to identify cells undergoing programmed cell death. The precision of this binding allows researchers to study these specific cells, making the technique a fundamental tool in cellular biology for investigating tissue development, disease, and the effects of therapeutic drugs.
Phosphatidylserine as a Cellular Signal
The plasma membrane of a cell is a barrier composed of a double layer of lipid molecules, known as the outer and inner leaflets. In a healthy, living cell, the distribution of these lipids is controlled and asymmetrical. Phosphatidylserine (PS) is a specific type of phospholipid that, under normal conditions, is found almost exclusively in the inner leaflet. This arrangement is actively maintained by specialized enzymes.
Proteins called “flippases” and “floppases” uphold this localization by using energy to move specific lipids between the two layers. Flippases transport PS from the outer leaflet to the inner one, ensuring it remains hidden from the external environment. This continuous process maintains the membrane’s integrity and is a hallmark of a viable cell.
When a cell receives signals to initiate apoptosis, one of the earliest changes is the breakdown of this membrane asymmetry. The enzymes keeping PS on the inner leaflet are inactivated, while enzymes known as “scramblases” become active. Scramblases rapidly move phospholipids between the leaflets, leading to the exposure of PS on the cell’s outer surface. This externalized PS acts as an “eat me” signal, prompting phagocytes to engulf the dying cell before it can release its contents and cause inflammation.
Annexin V as the Specific Detector
Annexin V’s utility in the laboratory comes from its high and specific affinity for phosphatidylserine. This binding is dependent on the presence of calcium ions, a feature scientists can control. Calcium causes a conformational change in the protein, enabling it to form a stable complex with exposed PS. In the absence of calcium, Annexin V will not bind, but when it is added, the protein rapidly attaches to its target.
This specific, calcium-dependent binding makes Annexin V an ideal probe for identifying apoptotic cells. Healthy cells keep their PS hidden on the inner membrane leaflet, so Annexin V cannot bind to them. It only attaches to cells that have begun apoptosis and flipped PS to their outer surface. This provides a clear marker for this early stage of cell death.
The Annexin V Staining Assay
The Annexin V staining assay is a laboratory procedure that uses the protein’s specific binding to phosphatidylserine. The process begins by collecting cells from a culture or tissue sample. These cells are then washed and suspended in a binding buffer containing the calcium ions necessary for Annexin V to function.
Next, a solution of Annexin V that has been chemically linked to a fluorescent molecule is added to the cells. Common fluorescent tags include fluorescein isothiocyanate (FITC), which glows green, or phycoerythrin (PE), which glows orange. The cells are incubated in this solution for 10 to 15 minutes, allowing the labeled Annexin V to bind to any cells with exposed PS.
After incubation, the stained cells are analyzed. One method is fluorescence microscopy, where a scientist can visually see individual glowing cells, indicating early apoptosis. A more common method is flow cytometry, a technique that passes thousands of cells per second through a laser beam to measure the fluorescence of each one. This analysis allows for the quantification of apoptotic cells within a large population.
Interpreting Staining Results
Data from an Annexin V assay is most informative when combined with a second stain that assesses cell membrane integrity. A viability dye such as Propidium Iodide (PI) or 7-AAD is used for this. These dyes cannot cross the intact membrane of a living cell and can only enter when the membrane has become permeable, which occurs in late-stage apoptosis or necrosis.
Using both Annexin V and a viability dye like PI allows researchers to distinguish several cell populations. Live, healthy cells will not be stained by either dye. Early apoptotic cells have exposed PS but an intact membrane, so they stain positive for Annexin V but negative for PI. Late-stage apoptotic or necrotic cells have lost membrane integrity, allowing both Annexin V to bind and PI to enter, resulting in a double-positive signal.
This dual-staining approach is visualized using a quadrant plot from a flow cytometer. The plot is divided into four sections, allowing for an accurate count of cells in different states:
- Lower-left quadrant: Live cells (Annexin V-/PI-)
- Lower-right quadrant: Early apoptotic cells (Annexin V+/PI-)
- Upper-right quadrant: Late apoptotic or necrotic cells (Annexin V+/PI+)
- Upper-left quadrant: Cellular debris
This detailed insight shows how a cell population is responding to a treatment or condition.