MTT Assay
Quick Facts
| Feature | Information |
|---|---|
| Category | Cell Viability / Cytotoxicity Assay |
| Principle | Tetrazolium Reduction |
| Detection Method | Colorimetric |
| Sample Type | Adherent and Suspension Cells |
| Quantitative | Yes |
| Typical Assay Time | 2-6 hours |
| Typical Output | Absorbance proportional to metabolically active cells |
Overview
The MTT Assay is one of the most widely used colorimetric assays for measuring cell viability, proliferation, and cytotoxicity. It is based on the ability of metabolically active cells to reduce the yellow tetrazolium salt MTT into insoluble purple formazan crystals.
Because only living cells possess sufficient metabolic activity to reduce MTT, the amount of formazan produced is proportional to the number of viable cells.
Biological Principle
The assay measures cellular metabolic activity, which serves as an indirect indicator of cell viability.
Viable cells contain active oxidoreductase enzymes, primarily within the mitochondria, that reduce MTT to insoluble formazan crystals. Cells that have lost metabolic activity produce little or no formazan.
Since metabolic activity may change independently of cell number, MTT measures metabolic competence rather than membrane integrity.
Principle of Detection
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a yellow tetrazolium compound.
Metabolically active cells reduce MTT to insoluble purple formazan crystals.
After incubation, the crystals are dissolved using an appropriate solubilization solution, and absorbance is measured using a microplate reader.
The intensity of the purple color is proportional to the metabolic activity of the cell population.
Workflow
Cells
↓
Add MTT Reagent
↓
Incubation
↓
Formazan Crystal Formation
↓
Solubilization
↓
Measure Absorbance
↓
Calculate Relative Cell Viability
What Does It Measure?
| Measurement | Interpretation |
|---|---|
| High absorbance | Greater metabolic activity |
| Low absorbance | Reduced metabolic activity |
| Increased signal | Higher viable cell population |
| Reduced signal | Cytotoxicity or reduced proliferation |
Applications
The MTT assay is commonly used for:
- Cell viability assessment
- Drug cytotoxicity screening
- Cell proliferation studies
- Cancer research
- Toxicology
- Chemotherapy sensitivity testing
- Biomaterial biocompatibility
- Dose-response analysis
Interpretation of Results
| Observation | Biological Interpretation |
|---|---|
| High absorbance | Large metabolically active cell population |
| Low absorbance | Reduced viability or metabolic inhibition |
| Progressive signal increase | Cell proliferation |
| Progressive signal decrease | Cytotoxicity or growth inhibition |
Advantages
- Simple and reliable
- Quantitative
- High-throughput compatible
- Cost-effective
- Widely validated
- Suitable for drug screening
Limitations
- Measures metabolic activity rather than direct cell number
- Endpoint assay
- Requires crystal solubilization before measurement
- Metabolic changes may occur without changes in viability
- Some compounds interfere with tetrazolium reduction or absorbance measurements
Comparison with Similar Assays
| Assay | Primary Measurement |
|---|---|
| MTT | Cellular metabolic activity |
| MTS | Cellular metabolic activity (soluble product) |
| XTT | Cellular metabolic activity (soluble product) |
| WST-1 | Cellular metabolic activity |
| CCK-8 | Cellular metabolic activity (water-soluble formazan) |
| Trypan Blue | Plasma membrane integrity |
| ATP Assays | Intracellular ATP concentration |
Common Misinterpretations
- Increased MTT signal does not necessarily indicate an increase in cell number.
- Reduced absorbance may reflect decreased metabolism rather than cell death.
- MTT cannot distinguish apoptosis from necrosis.
- Different cell types reduce MTT at different rates, making comparisons between unrelated cell lines inappropriate.
Key Takeaways
- The MTT assay is a colorimetric assay based on tetrazolium reduction by metabolically active cells.
- Living cells convert yellow MTT into insoluble purple formazan crystals.
- The amount of formazan produced correlates with cellular metabolic activity and is commonly used as an indirect measure of viability.
- MTT is widely used for cytotoxicity testing, proliferation studies, and drug screening.
- Because it measures metabolism rather than membrane integrity, results should be interpreted alongside complementary viability or apoptosis assays when necessary.
References
- Mosmann T. Rapid Colorimetric Assay for Cellular Growth and Survival. Journal of Immunological Methods (1983).
- Freshney RI. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications.
- Riss TL et al. Cell Viability Assays. Assay Guidance Manual.
- Thermo Fisher Scientific. MTT Cell Proliferation Assay Product Information.
- ATCC. Cell Proliferation and Viability Assays.