MTT Assay Protocol for Cell Viability and Proliferation

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About MTT Assay

Cytotoxicity assays are routinely used to calculate the effectiveness of a chemotherapeutic drug against cancer cells.

MTT assay is used to evaluate the cytotoxicity.

MTT assay evaluates the effect of a drug on the proliferative index of the cell population.

MTT Assay Mechanism

Principle of MTT Assay

In MTT assay, cells in the exponential phase of growth are exposed to a cytotoxic drug.

After addition of the drug, the cells are allowed to proliferate for two to three population-doubling times (PDTs).

This is a colorimetric assay that measures the reduction of yellow 3-(4,5- dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase.

The MTT dye enters into the cells and then passes into the mitochondria where it get reduced to an insoluble, coloured (dark purple) formazan product.

The cells are then solubilised with an organic solvent (eg. Isopropanol or DMSO) and the released, solubilised formazan reagent is measured by spectrophotometer.

Since reduction of MTT can only occur in metabolically active cells the level of activity is a measure of the viability of the cells.

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MTT Assay Requirements


1. Secondary cell line

2. Growth medium with 10% FCS; Growth medium with 5% FCS

3. Trypsin (0.25%) + EDTA, (1 mM) in PBS

4. MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma), 0.5 mg/ml, filter sterilized

5. Multiwell plates (96 well), Reservoir for multichannel pipette.

6. Pipettor tips, in an autoclavable tip box

7. Universal containers or tubes 8. Cytotoxic drug, 70% IPA


9. Plastic box (clear polystyrene, to hold plates)

10. Multichannel pipettor, neuber’s chamber

11. Dimethyl sulfoxide (DMSO)

12. ELISA plate reader

Procedure of MTT Assay

1. Split the cells.

2. Determine the optimal cell count for seeding in 96 well plates.

3. Seed optimum number of cells in 100 μl media (this number vary from cell to cell e.g. 4000 cells per well for MDA-MB-231 and 2000 cells per well for MCF-7 and SK-BR-3) in 96 well plates.

4. Incubate the plate for 18-24 hours in 5% CO2 incubator set at 370 C.

5. Treat the cells in triplicate when they reach at about 40-50% confluence. Use appropriate positive, negative and DMSO controls.

6. Incubate the plates in 5% CO2 incubator set at 370 C for 24, 48 or 72 hours depending on the desired time point for your experiment.

7. After the desired time point add 10 μl of MTT (5 mg/ml) each well in the media.

8. Incubate for 1-2 hours (depending on cell type) in 5% CO2 incubator set at 370 C until the formazan crystals formed that can be seen under the microscope.

9. Discard the media gently without disturbing formazan crystals.

10. Add 100-200 μl of DMSO per well to solubilise the crystals.

11. Incubate on shaker in dark for 10 minutes.

12. Read the absorbance at 540-595 nm.

MTT Assay plate
MTT Assay Citations


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