MTS 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 | 1-4 hours |
| Typical Output | Absorbance proportional to metabolically active cells |
| Readout | Soluble Formazan Dye |
Overview
The MTS Assay is a colorimetric assay used to assess cell viability, proliferation, and cytotoxicity by measuring cellular metabolic activity. It is based on the enzymatic reduction of the tetrazolium compound MTS into a water-soluble formazan product, eliminating the crystal dissolution step required in the MTT assay.
Because the formazan product is soluble in culture medium, the MTS assay is simpler, faster, and more suitable for high-throughput applications.
Biological Principle
Metabolically active cells contain oxidoreductase enzymes that reduce MTS into a soluble colored formazan product.
The amount of formazan produced is proportional to the metabolic activity of viable cells.
Like other tetrazolium assays, MTS measures metabolic competence rather than direct cell number or membrane integrity.
Principle of Detection
MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) is converted by viable cells into a water-soluble formazan dye.
An intermediate electron-coupling reagent, typically phenazine ethosulfate (PES), facilitates electron transfer during the reduction reaction.
The soluble formazan remains in the culture medium and can be measured directly without additional processing.
Workflow
Cells
↓
Add MTS Reagent
↓
Incubation
↓
Formation of Soluble Formazan
↓
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 MTS assay is commonly used for:
- Cell viability assessment
- Cytotoxicity testing
- Cell proliferation studies
- Drug discovery
- Cancer research
- Toxicology screening
- Biomaterial evaluation
- High-throughput compound screening
Interpretation of Results
| Observation | Biological Interpretation |
|---|---|
| High absorbance | High metabolic activity |
| Low absorbance | Reduced viability or metabolic inhibition |
| Increasing signal over time | Cell proliferation |
| Decreasing signal | Growth inhibition or cytotoxicity |
Advantages
- No crystal solubilization step
- Faster than MTT
- Simple one-step protocol
- High-throughput compatible
- Less handling reduces experimental variability
- Suitable for continuous plate-based workflows
Limitations
- Measures metabolic activity rather than direct cell number
- Endpoint assay
- Some compounds may chemically reduce MTS, producing false-positive results
- Colored media or test compounds may interfere with absorbance measurements
- Cell types differ in metabolic activity, affecting signal intensity
Comparison with Similar Assays
| Assay | Primary Measurement |
|---|---|
| MTS | Cellular metabolic activity (soluble formazan) |
| MTT | Cellular metabolic activity (insoluble formazan) |
| XTT | Cellular metabolic activity (soluble formazan) |
| WST-1 | Cellular metabolic activity (soluble formazan) |
| CCK-8 | Cellular metabolic activity (water-soluble formazan) |
| Trypan Blue | Plasma membrane integrity |
| ATP Assays | Intracellular ATP concentration |
Common Misinterpretations
- High absorbance does not necessarily indicate increased cell number.
- Reduced signal may result from metabolic inhibition rather than cell death.
- MTS cannot distinguish apoptosis from necrosis.
- Different cell types generate different amounts of formazan despite having similar cell numbers.
Key Takeaways
- The MTS assay is a colorimetric assay that measures cellular metabolic activity using a tetrazolium-based reaction.
- Unlike MTT, the reaction produces a water-soluble formazan dye, eliminating the need for a crystal dissolution step.
- The assay is simple, rapid, and well suited for high-throughput viability and cytotoxicity studies.
- MTS provides an indirect measure of viable cell number based on metabolic activity.
- Results should be interpreted alongside complementary assays when precise assessment of cell death mechanisms is required.
References
- Cory AH et al. Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Communications (1991).
- Riss TL et al. Cell Viability Assays. Assay Guidance Manual.
- Freshney RI. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications.
- Promega. CellTiter 96® AQueous One Solution Cell Proliferation Assay Technical Manual.
- ATCC. Cell Proliferation and Viability Assays.