The MTT Cell Viability and Proliferation Assay is employed to evaluate cellular metabolic activity as an indicator of cell viability, proliferation, and cytotoxicity. This colorimetric method relies on the conversion of a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT) to purple formazan crystals by metabolically active cells. These cells possess NAD(P)H-dependent oxidoreductase enzymes, which facilitate the reduction of MTT to formazan. Following this reaction, insoluble formazan crystals are dissolved using a solubilization solution, and the resulting colored solution is quantified by measuring absorbance at 500-600 nanometers using a multi-well spectrophotometer. A darker solution indicates a higher number of viable, metabolically active cells.

This protocol can be utilized for the following assessments:

  • Quantification of cell growth

  • Measurement of cytotoxicity (e.g., following anti-cancer drug treatments)

  • Study of cell activation

Moreover, since the MTT assay relies on the reduction of MTT to formazan crystals by metabolically active cells involving NAD(P)H-dependent oxidoreductase enzymes, which are present in the mitochondria, the protocol can also be used to assess mitochondrial functions.

Cell Viability and Proliferation Assay

  • 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).

  • Cell culture medium.

  • Phosphate-buffered saline (PBS).

  • Dimethyl sulfoxide (DMSO).

  • Plate reader.

Protocol:
 

1. Cell Seeding:

  • Seed cells in a 96-well microplate with a transparent bottom. Ensure cells are evenly distributed across each well. It's recommended to avoid to seed the cells in the perimeters wells due to the “edge effect”; in fact the perimeter wells are more exposed to the environment and tend to have different evaporation rates compared to the inner wells

2. Treatment Application:

  • Prepare the MTT stock solution at a concentration of 0.5 mg/ml in PBS. Store protected from light until use.

  • After the treatment period, carefully add 10 μl of the MTT solution to each well, ensuring not to dislodge the cells.

3. Incubation for Formazan Formation:

  • Place the microplate in a humidified incubator set at 37 °C with 5% CO₂ for 4 h. This allows for the reduction of MTT by metabolically active cells to form purple formazan crystals, visible under a microscope.

4. Removal of Medium and MTT Solution:

  • After incubation, carefully remove the cell medium and MTT solution from each well using a pipette, taking care not to disturb the formazan crystals at the bottom of the wells.

5. Washing:

  • Gently wash each well with PBS to remove any remaining medium and unreacted MTT, ensuring complete removal of PBS in subsequent steps.

6. Dissolving Formazan Crystals:

  • Add 100 μl of DMSO to each well to dissolve the formazan crystals. Cover the microplate with aluminum foil to protect from light.

7. Shaking:

  • Place the covered plate on an orbital shaker and shake gently at room temperature for 30 min to ensure complete solubilization of the formazan crystals. 

8. Measurement:

  • Measure the absorbance of each well using a microplate reader set at 590 nm. Ensure to complete the readings within 1 hour of solubilization to avoid precipitation.
     

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