MTT Cell Viability Assay Protocol: Metabolic Activity, Cytotoxicity and Analysis

The MTT assay is a colorimetric assay that measures reduction of the yellow tetrazolium salt MTT to purple formazan by metabolically active cells. It is widely used as a convenient proxy for cell viability, proliferation and cytotoxicity. However, MTT is fundamentally a metabolic activity assay: treatment can change cellular metabolism without changing cell number, and some compounds can interfere chemically with tetrazolium reduction or absorbance. Results should therefore be interpreted carefully and, for important conclusions, supported by an orthogonal viability or cell-counting method.

Equipment and reagents

• Healthy adherent or suspension cells.
• 96-well tissue-culture plate.
• MTT reagent.
• Culture medium appropriate to the cell line.
• Solubilisation reagent such as DMSO, acidified isopropanol or another validated solvent.
• Microplate reader.
• Untreated, vehicle and blank/no-cell controls.

Experimental design

• Establish a cell-number range that produces a linear MTT signal for the cell type.
• Use the same seeding density and culture duration across groups.
• Include a vehicle control at the same final solvent concentration used for treatment.
• Include wells containing medium + MTT but no cells to measure chemical/background absorbance.
• If the test compound is coloured, redox-active or particulate, run compound-only interference controls.

MTT assay procedure

1. Seed cells. Plate a density that will remain within the assay's linear range at the final readout time. Allow adherent cells to attach before treatment.
2. Apply treatment. Add compounds, cytokines or other experimental conditions. Include untreated and vehicle controls.
3. Prepare MTT reagent. A common starting solution is 5 mg/mL in PBS, sterile-filtered and protected from light. Follow the chosen reagent's instructions.
4. Add MTT. Introduce MTT at the validated final concentration. Published MTT workflows commonly prepare a concentrated MTT stock and dilute it into the culture medium, but the final concentration and added volume should be validated for the cell type, plate format and incubation time.
5. Incubate. Incubate at 37°C until sufficient purple formazan develops. Approximately 2–4 hours is common, but cell type and seeding density affect the required time.
6. Remove medium carefully. Avoid disturbing adherent cells or formazan crystals.
7. Solubilise formazan. Add DMSO, acidified isopropanol or the kit-specified solvent. Shake until crystals are fully dissolved.
8. Read absorbance. Common wavelengths are approximately 570–590 nm, often with a reference wavelength near 630–690 nm. Use the wavelength specified for the chemistry and reader.
9. Subtract blank signal. Correct all values for no-cell background.
10. Normalise. Express treated values relative to the matched untreated or vehicle control, provided all readings remain in the assay's linear range.

Example calculations

Relative metabolic activity (%) = 100 × corrected absorbance of treated sample / corrected absorbance of vehicle control.

This quantity is often reported as “cell viability”, but describe it as relative MTT/metabolic activity unless the relationship with viable cell number has been independently validated for the system.

Key limitations

• MTT reduction reflects cellular redox/metabolic state, not direct counting of living cells.
• Some treatments alter mitochondrial or cellular metabolism before inducing death.
• Compounds can absorb at the read wavelength or chemically reduce MTT.
• Very high cell densities can push the assay outside the linear range.
• Formazan solubilisation must be complete for reliable absorbance readings.

Troubleshooting

References and primary methods

1. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63. doi:10.1016/0022-1759(83)90303-4.
2. Berridge MV, Tan AS. Characterization of the cellular reduction of MTT: subcellular localization, substrate dependence, and involvement of mitochondrial electron transport. Arch Biochem Biophys. 1993;303:474–482. doi:10.1006/abbi.1993.1311.
3. Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev. 2005;11:127–152. doi:10.1016/S1387-2656(05)11004-7.
4. The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis. 2021. PMID:34884632.

Establishing the linear range before testing treatments

One of the most important MTT optimisation experiments is a cell-number titration. Seed a dilution series of the chosen cell type, keep incubation time constant and measure MTT signal. The useful range is the region where corrected absorbance increases predictably with cell number without plateauing. Treatment experiments should be designed so control wells remain inside this range at the final assay time.

MTT for cytotoxicity and dose-response studies

For drug-response experiments, use multiple concentrations spanning minimal effect through substantial loss of metabolic activity. Include enough biological replicates to estimate variability and keep vehicle concentration constant across the dilution series. An apparent IC50 derived from MTT is an IC50 for the MTT response under those conditions; it should not automatically be described as a concentration that kills 50% of cells unless that relationship has been independently demonstrated.

Compound-interference controls

Test compounds can interfere with MTT chemically or optically. Coloured compounds may absorb near the read wavelength, reducing agents can convert tetrazolium directly, and nanoparticles can scatter light. A useful interference plate contains medium + MTT + compound but no cells at each relevant concentration. If signal changes in the absence of cells, the assay is not reporting biology alone.

MTT versus alternative viability readouts

• Direct cell counting: measures cell number but not necessarily viability.
• ATP assays: highly sensitive metabolic viability readout, but also influenced by metabolic state.
• Resazurin assays: convenient and often non-destructive, but still redox-dependent.
• Membrane-integrity assays: LDH release or viability dyes provide a different mechanism and are valuable orthogonal controls.
• Clonogenic assays: measure long-term proliferative survival and can reveal effects missed by short-term metabolic assays.

Quality-control checklist

• No-cell blank is low and consistent.
• Vehicle control remains within the validated cell-number linear range.
• Replicate CV is acceptable for the experimental purpose.
• Formazan crystals are fully dissolved before reading.
• Readings are below the plate reader's saturation range.
• Test compounds do not generate cell-free interference at the assay wavelength.

Frequently asked questions

Is MTT a direct measure of viable cell number?

No. It measures cellular reduction of tetrazolium to formazan. That signal often correlates with viable cell number within a validated range but can change independently when metabolism is altered.

What wavelength should I use?

Common protocols use approximately 570–590 nm, often with a reference wavelength around 630–690 nm. Use the wavelength specified for the solvent and instrument configuration being used.

Why does my treatment reduce MTT signal without visible cell death?

The treatment may suppress mitochondrial or cellular redox activity, slow proliferation or alter cell size/metabolism. Confirm with a second assay that measures a different biological property.

Can I compare different cell lines using raw OD?

Not reliably. Cell lines can differ substantially in MTT reduction per cell. Build a separate cell-number relationship for each line and avoid equating raw absorbance with cell number across models.

Explore more with ABMIUM

If you are validating a drug or cytokine response, pair metabolic readouts with a mechanistic assay. Find your MATCH for apoptosis, proliferation or signalling antibodies, or explore flow cytometry, In-Cell ELISA and TUNEL.