What is the MTT Assay?
The MTT assay is a colorimetric assay for assessing cell metabolic activity. It is widely used in cancer research to measure the viability and proliferation of cancer cells. The assay involves the conversion of the yellow MTT tetrazolium salt to purple formazan crystals by metabolically active cells, a process facilitated by mitochondrial enzymes.
Why is the MTT Assay Important in Cancer Research?
The MTT assay is crucial in cancer research for several reasons. First, it provides a quantitative measure of cell viability, which is essential for evaluating the efficacy of anti-cancer drugs. Second, it is relatively simple, cost-effective, and can be performed in high-throughput formats, making it ideal for large-scale screening of potential chemotherapeutic agents.
Cells are plated in a 96-well plate and allowed to adhere and grow.
MTT solution is added to each well and incubated to allow for formazan crystal formation.
The medium is removed, and the crystals are dissolved in a solubilizing agent, typically DMSO.
The absorbance is measured using a spectrophotometer at a wavelength of around 570 nm.
The absorbance correlates with the number of viable cells, allowing for the calculation of cell viability percentages.
Simplicity and Cost-Effectiveness: The assay is easy to perform and does not require expensive reagents or equipment.
High Throughput: It can be adapted for use in 96-well or 384-well plates, enabling the screening of numerous samples simultaneously.
Reliability: The MTT assay provides consistent and reproducible results.
Non-Specificity: The assay measures overall metabolic activity, which may not be exclusively due to viable cancer cells.
Interference: Certain compounds may interfere with the MTT reduction process, leading to inaccurate results.
Formazan Solubility: The formazan crystals may be difficult to dissolve completely, affecting the absorbance readings.
How Does the MTT Assay Compare to Other Viability Assays?
The MTT assay is one of several assays used to measure cell viability, including the XTT, WST, and resazurin assays. Each has its own advantages and disadvantages. For example, the XTT and WST assays produce water-soluble formazan products, eliminating the need for a solubilizing step. However, the MTT assay remains popular due to its robustness and ease of use.
Optimizing the MTT Assay for Cancer Research
To obtain reliable and reproducible results, several factors must be optimized: Cell Density: The number of cells plated should be optimized to ensure a linear relationship between cell number and absorbance.
Incubation Time: The incubation time with MTT should be sufficient to allow for significant formazan formation but not so long as to cause cytotoxicity.
Solubilization: Complete dissolution of formazan crystals is essential for accurate absorbance readings.
Applications of the MTT Assay in Cancer Research
The MTT assay is used in various applications within cancer research: Drug Screening: It is widely used to screen potential chemotherapeutic agents for their cytotoxic effects on cancer cells.
Combination Therapy: The assay helps in evaluating the efficacy of combination therapies by measuring the synergistic effects of multiple drugs.
Mechanistic Studies: Researchers use the MTT assay to study the mechanisms of drug action and resistance in cancer cells.
Conclusion
The MTT assay is an indispensable tool in cancer research, offering a simple and effective method for assessing cell viability. Despite some limitations, its advantages make it a preferred choice for many applications, from drug screening to mechanistic studies. By optimizing the assay conditions, researchers can obtain reliable and reproducible data, contributing to the advancement of cancer therapies.
