Cell viability is the measure of the proportion of living and healthy cells within a population. Understanding a cell’s ability to maintain its basic functions is necessary for studying cell growth, death, and response to external stimuli. The MTT assay is a widely adopted, straightforward colorimetric method designed to quantify this viability. It is a foundational tool in toxicology, where the harmful effects of substances are tested, and in drug discovery, where the effectiveness of new therapeutic compounds is evaluated.
Measuring Metabolic Activity to Determine Viability
Researchers use a cell’s metabolic activity as a reliable proxy for its overall health. In the MTT assay, a viable cell is metabolically active and maintains a sufficiently reducing environment. Energy production pathways, particularly those involving the electron transport chain, generate reducing equivalents such as NADH and \(\text{NADPH}\), which are the indicators measured by the assay.
The recycling of these molecules by cellular enzymes reflects a cell’s ability to carry out essential biochemical reactions, including \(\text{ATP}\) synthesis. A healthy cell exhibits high metabolic function, while a cell suffering from toxicity shows a marked decrease. By measuring the capacity of a cell to perform a reduction reaction, the MTT assay provides a quantifiable snapshot of its physiological state.
The Chemical Transformation from MTT to Formazan
The core of the MTT assay relies on a specific chemical reaction involving the tetrazolium salt \(\text{MTT}\). This molecule is a pale yellow, water-soluble compound that easily passes through the cell membrane. Once inside, \(\text{MTT}\) encounters the cell’s reducing environment, specifically \(\text{NAD(P)H}\)-dependent oxidoreductase enzymes.
These enzymes, which include mitochondrial succinate dehydrogenase, catalyze the cleavage of the \(\text{MTT}\) tetrazolium ring. The chemical reduction transfers electrons from the cellular reducing equivalents to the \(\text{MTT}\) molecule. This transformation results in the formation of a distinct substance called formazan.
Formazan is an intensely purple-colored compound that is water-insoluble. Due to this insolubility, the newly formed formazan precipitates and accumulates as solid crystals within the cell’s cytoplasm. The amount of purple formazan produced is directly proportional to the concentration of active oxidoreductase enzymes and the number of metabolically viable cells. The intensity of the final purple coloration is the direct measure quantified by the assay.
Step-by-Step Procedure for the MTT Assay
The \(\text{MTT}\) assay begins with the careful preparation of the cells in a multi-well plate, typically in a 96-well format for high-throughput testing. Cells are cultured at a predetermined density and then test compounds, such as potential drugs or toxins, are applied at various concentrations. This initial incubation allows the cells to react to the treatment, which can last from several hours up to a few days, depending on the experimental design.
Reagent Addition and Incubation
Following the treatment phase, the \(\text{MTT}\) reagent, a sterile yellow solution, is added directly to the cell culture medium. The cells are then incubated, typically for one to four hours at \(37^\circ\text{C}\), to permit the uptake of the \(\text{MTT}\) and its enzymatic reduction to purple formazan crystals. Viable cells actively process the reagent, and the insoluble crystals form internally.
Solubilization
Because the formazan crystals are trapped inside the cells and are insoluble in the aqueous medium, a solubilization step must be performed. A solvent, such as dimethyl sulfoxide (\(\text{DMSO}\)) or a sodium dodecyl sulfate (\(\text{SDS}\)) solution, is added to each well. This solvent lyses the cells and dissolves the formazan into a homogenous, uniformly colored solution necessary for accurate measurement.
Spectrophotometric Measurement
The final step involves measuring the color intensity using a microplate reader, a specialized spectrophotometer. This instrument measures the absorbance, or optical density (\(\text{OD}\)), of the colored solution at a specific wavelength, typically 570 nanometers. A higher recorded \(\text{OD}\) value indicates a greater number of metabolically active cells.
Interpreting Data and Research Applications
The optical density (\(\text{OD}\)) values are used to calculate the cell viability percentage. Researchers normalize the absorbance readings from treated cells against an untreated control group, which represents 100% viability. This yields the percentage of viable cells remaining after exposure to the test compound. The data is then plotted to create a dose-response curve, illustrating the relationship between concentration and viability reduction.
A primary application of this data is calculating the \(\text{IC}_{50}\) value, or Inhibitory Concentration at 50%. The \(\text{IC}_{50}\) is the concentration of a substance required to reduce the viability of the cell population by 50%. This metric is a standard measure of a compound’s potency; a lower \(\text{IC}_{50}\) indicates a more potent inhibitory effect.
The MTT assay is heavily utilized in high-throughput screening during early-stage drug development, especially for identifying anti-cancer agents. Its ease of use and ability to process numerous samples simultaneously make it ideal for rapidly testing cytotoxic effects. The assay is also a standard tool in general toxicology studies to determine the concentration at which chemicals or pollutants begin to harm living cells.