The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is a commonly employed colorimetric technique for measuring cell viability, proliferation, and cytotoxicity. Recognized for its reliability, the MTT assay plays a crucial role in evaluating cellular health and metabolic function. It is widely utilized across various research domains, including drug development, cancer biology, toxicology, and tissue engineering.
By generating quantitative data, the assay enables researchers to analyze cellular responses to pharmaceuticals, toxins, and environmental stress, making it indispensable for studies involving apoptosis, necrosis, and metabolic changes. Nevertheless, careful optimization of experimental parameters is necessary to reduce artifacts and ensure accurate, reproducible results.
The mechanism of the MTT assay
The MTT assay is based on the ability of metabolically active cells to reduce the yellow MTT tetrazolium salt into insoluble purple formazan crystals. This color change serves as a visual and measurable indicator of cell viability. The reduction process is primarily driven by mitochondrial dehydrogenases—such as NAD(P)H-dependent oxidoreductases—which cleave the tetrazolium ring to produce formazan. Among these enzymes, succinate dehydrogenase plays a key role due to its involvement in mitochondrial electron transport. As the efficiency of this reduction reflects mitochondrial function, the assay offers a reliable assessment of cell viability.
However, MTT reduction is not limited to mitochondria. It can also occur in other cellular compartments, including the endoplasmic reticulum, lysosomes, and plasma membrane. As a result, the assay captures overall cellular metabolic activity and reducing capacity, rather than being exclusively mitochondrial.
After reduction, the generated formazan is solubilized and quantified spectrophotometrically. The amount of formazan formed correlates with the number of viable cells and their metabolic activity, making the MTT assay a sensitive method for detecting cytotoxic effects. Exposure to toxic substances or environmental stressors can impair enzymatic activity, leading to reduced formazan production and indicating compromised cell viability.
It is important to recognize that different cellular stressors may selectively impact various reduction pathways. Therefore, results from MTT assays should be interpreted in the context of the specific cellular environment and the nature of the applied stressors.
Common challenges and solutions
Low signal or weak absorbance readings
A frequent issue encountered in MTT assays is low signal intensity, which can result from suboptimal cell density, short incubation periods, or enzyme inactivity. It is important to seed the correct number of cells per well, usually 5,000 to 10,000 cells for adherent lines, such that enzymatic reduction of MTT yields a detectable formazan output.
Lack of proper incubation time or early addition of the reagents may also constrain crystal formation. Prolongation of the incubation period with MTT (typically 3–4 hours) increases the intensity of the signal. Additionally, verifying the freshness and storage conditions of MTT reagents is vital, as the compound is light-sensitive and prone to degradation.
High background noise or inconsistent results
High background absorbance may hide actual signals and decrease assay sensitivity. This can occur as a result of non-specific MTT reduction by dead cells or serum constituents, or due to insufficient cell culture media removal prior to the addition of the solubilizing reagent. To prevent this, media-only controls should be included, and the wash steps prior to the formazan solubilization step should be optimized. Moreover, it is always essential to ensure consistent timing across wells to avoid time-dependent signal variability.
Incomplete formazan solubilization
Poor dissolution of formazan crystals leads to erratic absorbance values and reduced sensitivity. To address this, solvents such as DMSO, isopropanol, or SDS in acidified isopropanol are commonly employed. Among these, DMSO is commonly preferred due to its ability to dissolve formazan rapidly and uniformly.
To ensure homogenous dissolution, gentle agitation of the microplate is recommended for 10–15 minutes following the addition of the solubilizing agent. If crystal clumping persists, the solvent volume may be increased, or mild sonication may be applied. However, vigorous shaking should be avoided, as it can lead to the detachment of loosely adherent cells.
Cell line-specific variability
Different cell lines metabolize MTT at varying rates depending on mitochondrial activity, doubling time, and metabolic profile. For instance, highly glycolytic cells like cancer cells may reduce MTT more actively than primary cells.
Therefore, it is essential to optimize assay conditions for each cell line, including seeding density, MTT concentration (usually 0.2–0.5 mg/mL), and incubation time. Pilot experiments are essential for establishing these parameters and avoiding misinterpretation of results.
Edge effects in microplates
Variations in temperature and humidity at the edges of 96-well plates can cause uneven evaporation, leading to inaccurate readings (commonly referred to as “edge effects”). To minimize this issue, the outermost wells should not be used for sample measurements or should be filled with sterile PBS or culture medium to maintain high humidity.
Additionally, the use of a plate sealer during incubation is recommended to stabilize environmental conditions and reduce variability across the plate.
Improving assay reliability
A variety of good laboratory practices can improve the validity of MTT assay results. These include using freshly prepared chemicals, assuring regular pipette calibration, technical replication, and having adequate positive and negative controls. Furthermore, normalization of MTT data in relation to blank or vehicle controls corrects for non-specific signals.
In high-throughput applications, automating liquid handling procedures and installing a plate washer can reduce human error and improve reproducibility. In chemical screening, pre-screening for interference with MTT reduction is vital, as several medications can directly reduce tetrazolium salts or inhibit mitochondrial enzymes regardless of cytotoxicity.
Alternatives and complementary assays
Although the MTT assay is reliable, it may not be optimal in all experimental conditions. Other assays, such as XTT, WST-1, or resazurin-based assays, have higher solubility or lower cytotoxicity and may be more suited to kinetic measurements or fragile cell lines. Combining a range of cell viability assays, such as LDH release for membrane integrity or ATP-based assays for energy metabolism, can provide a more comprehensive picture of cell responses to treatment.
