Using the MTT Assay: A Comprehensive Guide for Cell Viability Assessment

The MTT assay is a widely used colorimetric method for assessing cell viability and metabolic activity in various research fields. This article provides a step-by-step guide on how to perform the MTT assay, its principle, and the importance of selecting the appropriate tetrazolium salt. The information presented here is based on scientific literature and reputable sources in the field of cell biology.

Principle of the MTT Assay

The MTT assay measures the reduction of the yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye to an insoluble purple formazan product by mitochondrial succinate dehydrogenase. This reduction reaction occurs in viable cells and is dependent on the activity of cellular oxidoreductase enzymes.

Assessing Cell Viability

The reduction of MTT by cellular oxidoreductase enzymes reflects the number of viable cells present in the culture. The greater the formazan concentration, the higher the absorbance, indicating higher cell viability. The MTT assay is a reliable and quantitative method for evaluating cell viability and has been widely adopted in various experimental settings.

Tetrazolium Salts

MTT is the most commonly used tetrazolium salt in the assay. However, alternatives such as XTT, MTS, and WSTs are also available. These tetrazolium salts offer different absorption spectra and have varying solubility properties, providing flexibility in experimental design and data interpretation.

Assay Procedure

The MTT assay is typically performed in a 96-well plate format. The following steps outline the general procedure:

1. Cell Seeding: Seed the cells of interest in a 96-well plate and allow them to adhere and grow.
2. MTT Solution Addition: Add the MTT solution to each well, ensuring proper distribution.
3. Formazan Solubilization: After a specific incubation period, remove the culture medium and add a solubilization solution to dissolve the formazan crystals.
4. Absorbance Measurement: Measure the absorbance of the formazan product at a wavelength between 500 and 600 nm using a spectrophotometer.
5. Calculation of Cell Viability: The amount of formazan produced is directly proportional to the number of living cells present in the culture. Calculate the percentage of cell viability using the formula: Viability % = 100 x OD550e/OD550b, where OD550e is the mean value of the measured optical density of the test item, and OD550b is the mean value of the measured optical density of the negative control.

Solubilization Solution

The original method for solubilizing the formazan crystals involves using acidified isopropanol. The acidic pH helps avoid interference with recording absorbance. However, it is important to note that organic solvents may precipitate proteins from serum-supplemented culture medium, causing light scattering. Researchers should consider alternative solubilization methods based on their specific experimental requirements.

It is worth mentioning that additional factors, such as incubation time and temperature, may vary depending on the specific experimental conditions and cell type used. Researchers should optimize these parameters to ensure reliable and reproducible results.

By following these guidelines, researchers can effectively use the MTT assay to assess cell viability, providing valuable insights into cellular metabolic activity and facilitating various areas of scientific research.

Sources:
– ScienceDirect
– Wikipedia
– Promega

FAQs

What is the principle behind the MTT assay?

The MTT assay measures the reduction of the yellow MTT dye to a purple formazan product by mitochondrial succinate dehydrogenase. The reduction reaction occurs in viable cells and reflects their metabolic activity.

How does the MTT assay assess cell viability?

The reduction of MTT by cellular oxidoreductase enzymes reflects the number of viable cells present. The higher the concentration of formazan, the greater the absorbance, indicating higher cell viability.

What are the different tetrazolium salts used in the MTT assay?

MTT is the most commonly used tetrazolium salt in the assay. However, alternatives such as XTT, MTS, and WSTs are also available. These salts have different absorption spectra and solubility properties, providing flexibility in experimental design.

What are the general steps involved in performing the MTT assay?

The general procedure of the MTT assay involves cell seeding, addition of MTT solution, formazan solubilization, absorbance measurement, and calculation of cell viability.

What solubilization solution is typically used in the MTT assay?

The original method uses acidified isopropanol to solubilize the formazan crystals. However, organic solvents may precipitate proteins from serum-supplemented culture medium, causing light scattering. Researchers should consider alternative solubilization methods based on their specific experimental requirements.

How is the absorbance of the formazan product measured?

The absorbance of the formazan product is usually measured at a wavelength between 500 and 600 nm using a spectrophotometer. This measurement provides quantitative data on cell viability.

How is cell viability calculated in the MTT assay?

The amount of formazan produced is directly proportional to the number of living cells present in the culture. Cell viability is calculated using the formula: Viability % = 100 x OD550e/OD550b, where OD550e is the mean value of the measured optical density of the test item, and OD550b is the mean value of the measured optical density of the negative control.

Are there any additional factors to consider when using the MTT assay?

Yes, factors such as incubation time, temperature, and optimization of assay parameters may vary depending on the specific experimental conditions and cell type used. Researchers should carefully consider these factors to ensure reliable and reproducible results.