In-Vitro Acid Phosphatase for Cell Viability Assay

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 of Work

• Traditionally, the in vitro determination of toxic effects of unknown compounds or nanoparticles has been performed by counting viable cells after staining with a vital dye. 
• Alternative methods used are a measurement of radioisotope incorporation as a measure of DNA synthesis, counting by automated counters and others that rely on dyes and cellular activity. 
• The acid phosphatase assay is a means of measuring the mass of cells via cell-membrane associated acid phosphatase. 
• The acid phosphatase cell viability assay method is simple, accurate, and yields reproducible results. 
• The key component in cell viability assay is p-Nitrophenyl phosphate. 
• Solutions of p-Nitrophenyl phosphate in medium or PBS without phenol red are colourless.

• Cell-membrane associated acid phosphatase cleaves the p-Nitrophenyl phosphate substrate yielding a yellow-coloured compound (p-Nitrophenol) which is soluble in aqueous solutions. 
• The resulting yellow solution is measured spectrophotometrically. 
• An increase or decrease in cell numbers results in a simultaneous change in the amount of substrate converted, indicating the degree of cytotoxicity caused by the tested nanomaterial.