In Vitro Cell Viability by the Lactate Dehydrogenase Test Assay (LDH)

Theoretical Background / Context

• Cytotoxicity is the quality of being toxic to cells.
•  Lactate dehydrogenase 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.

Lactate Dehydrogenase Assay Principle of Work

• Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme that is found in all cells. 
• LDH is rapidly released into the cell culture supernatant when the plasma membrane is damaged, a key feature of cells undergoing apoptosis, necrosis, and other forms of cellular damage through lactate dehydrogenase release assay.
• LDH activity can be easily quantified by using the NADH produced during the lactate dehydrogenase reaction conversion of lactate to pyruvate to reduce a second compound in a coupled reaction into a product with properties that are easily quantitated. 

• The lactate dehydrogenase assay protocol measures the reduction of a yellow tetrazolium salt, INT, by NADH into a red, water-soluble Formazan-class dye by absorbance at 492 nm based on the ldh assay principle.
• The amount of formazan is directly proportional to the amount of LDH in the culture, which is, in turn, directly proportional to the number of dead or damaged cells.