In-Vitro Annexin V Assay Binding/Propidium Iodide Uptake

Theoretical Background / Context

• Cytotoxicity is the quality of being toxic to cells. Cytotoxicity assays are widely used by the pharmaceutical industry to screen for cytotoxicity in compound libraries. 
• Researchers, as in Nanotechnology, can either look for cytotoxic nano-based materials, if they are interested in developing a nanomedicine that targets rapidly dividing cancer cells, for instance; or they can screen "hits" from initial high-throughput nanoparticle screens for unwanted cytotoxic effects before investing in their development as a nanomedicine. 
• Assessing cell membrane integrity is one of the most common ways to measure cell viability and cytotoxic effects. Compounds that have cytotoxic effects often compromise cell membrane integrity. Vital dyes, such as trypan blue or propidium iodide are normally excluded from the inside of healthy cells; however, if the cell membrane has been compromised, they freely cross the membrane and stain intracellular components. 
• Alternatively, membrane integrity can be assessed by monitoring the passage of substances that are normally sequestered inside cells to the outside. 
• Protease biomarkers have been identified that allow researchers to measure relative numbers of live and dead cells within the same cell population. 
• The live-cell protease is only active in cells that have a healthy cell membrane and loses activity once the cell is compromised and the protease is exposed to the external environment. 
• The dead-cell protease cannot cross the cell membrane, and can only be measured in culture media after cells have lost their membrane integrity. 
• Cytotoxicity can also be monitored by measuring the reducing potential of the cells using a colourimetric reaction, or using ATP content as a marker of viability. 
• Such ATP-based assays include bioluminescent assays in which ATP is the limiting reagent for the luciferase reaction. 
• A label-free approach to follow the cytotoxic response of adherent animal cells in real-time provides the kinetics of the cytotoxic response rather than just a snapshot like many colourimetric endpoint assays.

Principle Work of Annexin V Assay

In the Annexin V assay:

• The surface of healthy cells is composed of lipids that are asymmetrically distributed on the inner and outer leaflet of the plasma membrane. 
• One of these lipids, phosphatidylserine (PS), is normally restricted to the inner leaflet of the plasma membrane and is, therefore, only exposed to the cell cytoplasm. 

• However, during apoptosis lipid asymmetry is lost and PS becomes exposed on the outer leaflet of the plasma membrane through phosphatidylserine externalization.
• Annexin V, a 36-kDa calcium-binding protein, binds to PS; therefore, fluorescently labelled Annexin V can be used to detect PS that is exposed on the outside of apoptotic cells in the annexin assay apoptosis.

• Annexin V staining can also stain necrotic cells because these cells have ruptured membranes that permit Annexin V to access the entire plasma membrane in annexin v apoptosis detection.
• However, apoptotic cells can be distinguished from necrotic cells by co-staining with propidium iodide (PI) because PI enters necrotic cells but is excluded from apoptotic cells, enabling necrosis detection.