In-Vitro Neutral Red Uptake 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 such as neutral red uptake assay.

Neutral Red Assay Principle of Work

• The neutral red uptake assay provides a quantitative estimation of the number of viable cells in culture. 
• It is one of the most used cytotoxicity tests with many biomedical and environmental applications. 
• It is based on the ability of viable cells to incorporate and bind the supravital dye virtual neutral red in the lysosomes.

• Most primary cells and cell lines from diverse origins may be successfully used in neutral red uptake assay.
• Cells are seeded in 96-well tissue culture plates and are treated for the appropriate period. 
• The plates are then incubated for 3h with a medium containing neutral red. 
• The cells are subsequently washed, the dye is extracted in each well, and the absorbance is read using a spectrophotometer. 
• The assay can be completed in less than 4 hours.