MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent is a crucial tool used in cell biology research to assess cell metabolic activity through the measurement of the reduction of MTT to its insoluble formazan product. This article provides an overview of MTT reagent, its purpose, measurement techniques, related tetrazolium salts, solubilization methods, and its significance in cell viability assessment.
The primary purpose of MTT reagent is to evaluate the metabolic activity of cells by detecting their ability to reduce MTT. This reduction process is catalyzed by NAD(P)H-dependent cellular oxidoreductase enzymes and reflects the number of viable cells present in a culture.
MTT reagent is initially in a yellow form and undergoes a color change when reduced to a purple formazan product in living cells. The intensity of the purple color formed is directly proportional to the metabolic activity of the cells.
The amount of formazan produced during the MTT assay is directly related to the number of living cells present in the culture. The measurement of formazan can be quantified by assessing the absorbance at a specific wavelength, typically between 500 and 600 nm, using a spectrophotometer.
MTT is classified as a tetrazolium salt, and there are several related tetrazolium salts used in conjunction with different electron acceptors. These include XTT, MTS, and WSTs. Each of these salts has specific advantages and applications in cell viability assessments.
After the MTT assay, the insoluble purple formazan product needs to be dissolved into a colored solution for accurate measurement. Solubilization is typically achieved using a solubilization solution, such as dimethyl sulfoxide or an acidified ethanol solution. This step ensures that the formazan product is in a soluble form for absorbance measurement.
Sensitivity and Dynamic Range
XTT has been proposed as an alternative to MTT due to its higher sensitivity and dynamic range. XTT eliminates the need for a solubilization step, as the formazan dye it produces is water-soluble, offering enhanced convenience and accuracy in cell viability assessments.
In addition to XTT, other water-soluble tetrazolium salts such as MTS and WSTs have been developed. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) can be used as a one-step MTT assay, simplifying the process. WSTs offer different absorption spectra of the formed formazans and provide direct reading, increased signal effectiveness, and reduced toxicity to cells compared to MTT.
Mechanism of Reduction
Reduction of tetrazolium dyes, including MTT, is generally assumed to be dependent on NAD(P)H-dependent oxidoreductase enzymes in the cytosolic compartment of the cell. However, there is evidence of spontaneous MTT reduction occurring in lipidic cellular structures without enzymatic catalysis. This indicates alternative pathways for formazan formation in addition to enzymatic reduction.
The MTT assay serves as a valuable tool for assessing cell viability by measuring the metabolic activity of cells. It is important to note that assay conditions can influence metabolic activity without necessarily affecting cell viability. Therefore, proper controls and experimental design are essential for accurate interpretation of MTT assay results.
The MTT assay protocol typically involves the following steps:
- Inoculating cells into a 96-well plate
- Adding MTT solution to the wells
- Incubating the plate to allow the reduction of MTT by viable cells
- Removing the medium and adding a solubilization solution to dissolve the formazan crystals
- Measuring the absorbance of the colored solution at a specific wavelength using a spectrophotometer
By following this protocol, researchers can obtain reliable data on cell metabolic activity and make accurate assessments of cell viability using MTT reagent.
1. ScienceDirect Topics. MTT Assay – an overview. Retrieved from: https://www.sciencedirect.com/topics/neuroscience/mtt-assay
2. Wikipedia. MTT assay. Retrieved from: https://en.wikipedia.org/wiki/MTT_assay
3. Abcam. MTT assay protocol. Retrieved from: https://www.abcam.com/kits/mtt-assay-protocol
What is the purpose of MTT reagent?
MTT reagent is used to assess cell metabolic activity by measuring the reduction of MTT to its insoluble formazan product.
How does MTT reagent change color during the assay?
MTT reagent starts as a yellow substance and is reduced to a purple formazan product in living cells.
How is the amount of formazan produced related to cell number?
The quantity of formazan produced is directly proportional to the number of living cells in the culture. It can be quantified by measuring the absorbance at a specific wavelength, typically between 500 and 600 nm.
Are there other tetrazolium salts similar to MTT?
Yes, there are related tetrazolium salts such as XTT, MTS, and WSTs that can be used in conjunction with different electron acceptors.
How is the insoluble formazan product solubilized?
After the MTT assay, the insoluble purple formazan product is dissolved into a colored solution using a solubilization solution, such as dimethyl sulfoxide or an acidified ethanol solution.
Is there a more sensitive alternative to MTT reagent?
XTT has been proposed as a replacement for MTT due to its higher sensitivity and dynamic range. It also eliminates the need for a solubilization step.
Are there water-soluble alternatives to MTT?
Yes, MTS and WSTs are water-soluble tetrazolium salts that offer advantages over MTT, including direct reading, higher signal effectiveness, and decreased toxicity to cells.
What is the mechanism of reduction for MTT?
Reduction of tetrazolium dyes, including MTT, is generally assumed to be dependent on NAD(P)H-dependent oxidoreductase enzymes in the cytosolic compartment of the cell. However, there is evidence of spontaneous MTT reduction in lipidic cellular structures without enzymatic catalysis.