What is the MTT Assay?
The MTT assay is a colorimetric assay for assessing cell metabolic activity. It is based on the reduction of the yellow tetrazolium salt MTT to purple formazan crystals by mitochondrial dehydrogenase enzymes in living cells. This biochemical reaction occurs only in viable cells, making the MTT assay a useful method for measuring cell viability and cytotoxicity.
How Does the MTT Assay Work?
Cells are incubated with the MTT reagent, where metabolically active cells convert MTT into insoluble formazan crystals. These crystals are then solubilized, and the resulting colored solution is quantified by measuring its absorbance with a spectrophotometer. The absorbance value is directly proportional to the number of viable cells.
Applications in Toxicology
In toxicology, the MTT assay is widely used to evaluate the cytotoxic effects of various substances, such as chemicals, drugs, and environmental pollutants, on cultured cells. It helps in determining the IC50 value, which is the concentration of a substance that inhibits 50% of cell viability. This data is crucial for risk assessment and regulatory purposes.Advantages of the MTT Assay
The MTT assay offers several advantages: Simple and cost-effective
High-throughput capability
Quantitative results
Non-radioactive and safe
Limitations of the MTT Assay
Despite its advantages, the MTT assay has limitations: Interference by certain compounds that can reduce MTT directly
Cannot distinguish between cell death mechanisms (e.g., apoptosis, necrosis)
Not suitable for non-adherent cells without additional modifications
Protocol for MTT Assay
A standard protocol for performing the MTT assay involves several key steps: Culture cells in a 96-well plate until they reach the desired confluency.
Treat cells with test substances for a specified period.
Add MTT reagent to each well and incubate for 3-4 hours at 37°C.
Remove the medium and dissolve the formazan crystals with a solubilization solution (e.g., DMSO).
Measure the absorbance at 570 nm using a microplate reader.
Interpretation of Results
The absorbance readings obtained from the MTT assay are used to calculate cell viability. A decrease in absorbance indicates reduced cell viability, suggesting cytotoxicity of the test substance. Conversely, an increase or no change in absorbance suggests that the cells are not affected or are proliferating.Alternative Assays
While the MTT assay is popular, there are alternative assays for measuring cell viability and cytotoxicity, including: MTT-derived assays like the MTS and XTT assays
Resazurin-based assays (e.g., Alamar Blue)
ATP-based assays (e.g., CellTiter-Glo)
These alternatives may offer advantages in terms of sensitivity, dynamic range, and compatibility with different cell types.
Conclusion
The MTT assay remains a cornerstone in toxicology for evaluating the cytotoxicity of various substances. Its simplicity, cost-effectiveness, and high-throughput capability make it a valuable tool in both research and regulatory settings. However, careful consideration of its limitations and potential interferences is essential for accurate interpretation of results.
