The MTT assay, formally known as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, stands as a widely accepted colorimetric method in cellular biology. This technique provides a quantitative assessment of cell health, serving as a standard tool for measuring cell metabolic activity, proliferation rates, and the cytotoxic effects of various compounds. Developed in 1983, it offered researchers a non-radioactive, high-throughput alternative to earlier, more cumbersome cell viability tests. The assay is instrumental across diverse scientific disciplines, including drug discovery, toxicology screening, and cancer research, where understanding cellular responses to external stimuli is paramount.
The Principle: What the Assay Measures
The underlying principle of the MTT assay is not a direct count of every living cell but rather a measure of the cell population’s overall metabolic competence. This approach assumes that a metabolically active cell is also a viable cell, making the assay a reliable indicator of health and proliferative capacity. The test relies entirely on the functional integrity of the cellular machinery responsible for energy production.
Specifically, the assay probes the activity of certain dehydrogenases, which are enzyme complexes found predominantly within the mitochondria of eukaryotic cells. These enzymes participate in the cellular respiration process, which is the mechanism by which a cell generates energy. A healthy, actively growing cell possesses a high level of this enzymatic activity, signaling robust metabolic function.
Cells that are non-viable, dying, or severely damaged exhibit significantly reduced or absent dehydrogenase activity. Consequently, they do not participate in the chemical conversion that the assay is designed to detect. The output of the assay is thus a reflection of the collective reducing power of the cell population, which correlates strongly with the number of viable cells present in the sample.
The Core Chemical Reaction
The observable change in the MTT assay results from a specific molecular transformation driven by the cell’s active metabolism. The assay begins with the addition of the MTT reagent, which is a pale yellow tetrazolium salt that is soluble in water. This colorless substrate diffuses across the cell membrane and enters the intracellular space, where the reaction takes place.
Inside the metabolically active cells, the tetrazolium ring structure of the MTT substrate is cleaved. This reduction reaction is primarily catalyzed by the enzyme succinate dehydrogenase, a component of the mitochondrial electron transport chain. Other cellular reductases, including those dependent on the coenzymes \(\text{NAD}(\text{P})\text{H}\), also contribute to this process by providing the electrons necessary to drive the chemical conversion.
The acceptance of electrons results in the formation of a product known as formazan. Unlike the initial water-soluble yellow MTT, formazan forms as dark purple crystals that are insoluble in aqueous solutions. These crystals precipitate and accumulate within the reducing environment of the cell.
Because the formazan product is insoluble, an additional step is required to dissolve the crystals before they can be quantified. The intensity of the purple color observed in the final solution is directly proportional to the amount of formazan produced, which reflects the population of metabolically active cells.
Step-by-Step Laboratory Procedure
Performing the MTT assay involves a series of controlled laboratory steps designed to translate the cellular metabolic activity into a measurable optical signal.
Cell Preparation and Treatment
The procedure typically begins with the preparation of cells, which are plated at a specific density into multi-well plates. Cells are subsequently treated with the test compounds, such as potential drug candidates, for a defined period, often ranging from 24 to 72 hours. This initial incubation phase allows the treatment to exert its effect on the cells.
MTT Incubation
Following the treatment period, the yellow MTT reagent is added directly to the culture medium in each well, typically reaching a final concentration of around 0.5 \(\text{mg/mL}\). The plates are then returned to the incubator for a second, shorter incubation period, usually between one and four hours. This time allows the viable cells to actively internalize the MTT and perform the enzymatic reduction to the insoluble formazan crystals.
Solubilization and Measurement
After this second incubation, the culture medium containing the remaining yellow MTT must be carefully removed, leaving the purple formazan crystals lodged within the cells at the bottom of the well. A solubilization agent, such as dimethyl sulfoxide (\(\text{DMSO}\)) or an acidified alcohol solution like isopropanol, is then added to each well. The purpose of this step is to dissolve the insoluble purple formazan crystals, transforming them into a homogeneous, measurable solution. To ensure complete dissolution and uniform color distribution, the plate is typically agitated gently on an orbital shaker for a short time. The final, colored solution is then quantified using a specialized instrument called a microplate reader, or spectrophotometer. This instrument measures the optical density (\(\text{OD}\)) of the solution, which is the final data point used for analysis.
Interpreting Results and Data Output
The final step in the MTT assay is to interpret the light absorbance measurements captured by the microplate reader. The spectrophotometer is set to measure the optical density (\(\text{OD}\)) of the purple formazan solution, with the optimal wavelength for detection generally centered at 570 \(\text{nm}\). A secondary reference wavelength, such as 630 \(\text{nm}\) or 650 \(\text{nm}\), is often used to subtract background noise caused by things like plate imperfections or residual medium components, ensuring a more accurate reading.
A fundamental assumption in the interpretation is that the intensity of the purple color, and thus the measured absorbance value, is directly proportional to the quantity of formazan produced. Since formazan production is tied to metabolic activity, a higher absorbance value signifies a greater number of viable, metabolically active cells. Conversely, a lower absorbance indicates decreased cell viability or proliferation.
Researchers normalize the data by comparing the absorbance values from the treated wells to those from untreated control wells, which represent 100% viability. This comparison allows for the calculation of the percentage of cell viability or cytotoxicity using a simple ratio formula. The resulting data is frequently plotted as a dose-response curve, illustrating the effect of increasing concentrations of a test compound on cell viability. From this curve, researchers can calculate the \(\text{IC}_{50}\) value, which is the concentration of the substance required to inhibit cell growth or cause cell death in 50% of the cell population.