The MTT assay is one of the most widely used methods for evaluating anticancer activity in research and drug development. It is a colorimetric assay that measures cell viability and proliferation, providing critical insights into the effectiveness of potential cancer treatments.
This topic explores the principle of the MTT assay, its applications in anticancer studies, and the step-by-step procedure used in laboratories worldwide.
What is the MTT Assay?
Definition and Overview
The MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a cytotoxicity assay used to assess cell metabolic activity. It determines cell viability based on the ability of living cells to convert MTT into formazan, a purple-colored product that can be quantified using a spectrophotometer.
Key Applications
- Screening anticancer drugs to determine their effectiveness.
- Measuring cell proliferation in different biological experiments.
- Assessing cytotoxicity of chemicals and natural compounds.
- Testing the effects of radiation or genetic modifications on cell survival.
Principle of the MTT Assay
How the Assay Works
The MTT assay is based on cellular metabolism. Living cells contain mitochondrial dehydrogenase enzymes, which reduce the yellow MTT compound to purple formazan crystals. The more viable cells in a culture, the more formazan is produced, resulting in a higher absorbance value.
MTT Reduction Process
- MTT enters the cell membrane.
- Mitochondrial enzymes reduce MTT to formazan.
- Formazan accumulates inside cells, producing a purple color.
- A solubilizing agent dissolves formazan for spectrophotometric analysis.
Importance in Anticancer Research
Cancer cells treated with anticancer agents exhibit reduced metabolic activity, leading to lower MTT conversion. This decrease in formazan formation allows researchers to determine drug-induced cytotoxicity.
Step-by-Step Protocol for MTT Assay
Materials Required
- MTT reagent (prepared in PBS or media)
- Cell culture media
- 96-well plate
- Anticancer drug solutions
- Dimethyl sulfoxide (DMSO) or acidified isopropanol
- Spectrophotometer (ELISA reader)
Procedure
1. Cell Seeding
- Plate cancer cells (e.g., HeLa, MCF-7, A549) into a 96-well plate.
- Incubate at 37°C with 5% CO₂ for 24 hours to allow adhesion.
2. Drug Treatment
- Add different concentrations of the anticancer drug to the wells.
- Incubate for 24-72 hours to allow drug action.
3. Adding MTT Reagent
- Prepare MTT solution (5 mg/mL in PBS).
- Add 10-20 µL of MTT solution to each well.
- Incubate for 3-4 hours to allow formazan formation.
4. Formazan Solubilization
- Remove the media carefully without disturbing crystals.
- Add DMSO (100 µL per well) to dissolve formazan.
- Incubate for 10-15 minutes at room temperature.
5. Absorbance Measurement
- Read absorbance at 570 nm using a microplate reader.
- Compare readings between treated and control groups to evaluate cytotoxicity.
Interpreting MTT Assay Results
Cell Viability Calculation
The percentage of viable cells is determined using the formula:
A lower viability percentage indicates higher cytotoxicity, suggesting effective anticancer activity.
IC₅₀ Determination
- The IC₅₀ (half-maximal inhibitory concentration) is the drug concentration that reduces cell viability by 50%.
- A low IC₅₀ value means high potency, making the compound a promising anticancer agent.
Advantages of the MTT Assay
1. High Sensitivity
- Detects low levels of cell activity, making it ideal for screening anticancer drugs.
2. Rapid and Cost-Effective
- Requires minimal reagents and time, making it an affordable cytotoxicity test.
3. Reliable and Reproducible
- Produces consistent results across multiple experiments.
4. Versatile for Different Cell Types
- Can be used for adherent and suspension cells in various cancer models.
Limitations of the MTT Assay
1. Interference with Metabolic Inhibitors
- Some anticancer drugs interfere with mitochondrial function, affecting MTT reduction.
2. Non-Specificity
- Measures overall metabolic activity, not direct cell death.
3. Incomplete Formazan Solubilization
- Poor solubilization may lead to inconsistent readings.
To overcome these issues, researchers sometimes use alternative assays like:
- XTT or MTS assays (more soluble derivatives).
- Live/dead staining to differentiate apoptosis from necrosis.
Comparison with Other Cytotoxicity Assays
| Assay Type | Detection Method | Advantages | Limitations |
|---|---|---|---|
| MTT Assay | Formazan absorbance | High sensitivity, easy to use | Requires solubilization step |
| XTT Assay | Water-soluble dye | No need for solubilization | Less stable than MTT |
| MTS Assay | Tetrazolium salt reduction | Faster than MTT | Can be less sensitive |
| Trypan Blue Exclusion | Cell membrane integrity | Simple and direct | Not as quantitative |
| Annexin V Staining | Apoptosis detection | Specific for programmed cell death | Requires flow cytometry |
Real-World Applications of MTT Assay in Cancer Research
1. Evaluating Chemotherapy Drugs
- The MTT assay helps test drug efficacy against various cancers, including breast, lung, and colon cancer.
2. Screening Natural Compounds
- Many plant-derived extracts (e.g., curcumin, resveratrol) are tested for anticancer properties.
3. Studying Drug Resistance
- Researchers use MTT to investigate why some cancer cells resist chemotherapy.
4. Assessing Combination Therapies
- The assay helps determine if drug combinations enhance cytotoxic effects.
The MTT assay is a powerful tool for assessing anticancer activity in laboratory settings. Its ability to measure cell viability, drug efficacy, and cytotoxicity makes it an essential method in cancer research and drug discovery.
Despite some limitations, its simplicity, reliability, and cost-effectiveness ensure its continued use in testing new cancer treatments. Researchers worldwide rely on the MTT assay to identify promising anticancer agents that may one day lead to effective cancer therapies.