In-Vitro Viability Assay Using Tetrazolium Salt XTT

Theoretical Background / Context

• Cytotoxicity is the quality of being toxic to cells. 
• XXT 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.

Working Principle of the XTT Assay Protocol

• The mitochondria in the cellular cytoplasm have many enzymes. 
• Among these, a specific enzyme system named the ‘succinate-tetrazolium reductase' system belongs to the respiratory chain of the mitochondria and is only active in viable cells. 
• This XTT cell viability assay experiment aims to test the activity of such an enzymatic system and measures this activity in nanoparticles-treated cells in comparison with the control untreated cells.
• The use of tetrazolium salts, including XTT (2, 3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbox-anilide), to assay cell proliferation, cell viability, and/or cytotoxicity is a widespread, established practice. 
• Cleavage of the tetrazolium salt to formazan occurs via the succinate-tetrazolium reductase system in the mitochondria of metabolically active cells.

• The reaction is attributed mainly to mitochondrial enzymes and electron carriers, but a number of other non-mitochondrial enzymes have been implicated. 

• XTT, a yellow tetrazolium salt, is cleaved to a soluble orange formazan dye, which can be measured by absorbance at 490 (or 450) nm in a microplate reader.