MTT Assay Protocol

Licia Miller   Product Manager

MTT colorimetry is a detection method based on cell metabolic activity. It is widely used in cell biology and pharmacology research to evaluate cell proliferation, cell activity, cytotoxicity, and drug screening.

The core principle of the MTT colorimetric assay is to use the succinate dehydrogenase in the mitochondria of living cells to reduce exogenous MTT (3- (4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide) (yellow) to a water-insoluble blue-purple crystal-formazan. Dead cells cannot form formazan crystals due to the lack of this reducing ability. The number of living cells can be indirectly reflected by adding dimethyl sulfoxide (DMSO) to dissolve the formazan crystals and measuring the light absorption value (OD value) at a wavelength of 490 nm or 570 nm on a microplate reader. Within a certain range of cell numbers, the amount of formazan crystals formed is proportional to the number of living cells.

Phase 1   Preparation of MTT solution

MTT is soluble in water, ethanol, buffered saline solutions, and culture media. Generally, we recommend using PBS solvent to prepare a 5 mg/mL MTT solution.

1. Dissolve MTT in PBS to prepare 5 mg/mL MTT solution.

2. Use vortexing or sonication to ensure that the MTT is completely dissolved.

3. Sterilize by filtration using a 0.22 µm filter.

4. After the MTT solution is divided, store it at -20℃ (it can be stored stably for at least 6 months).

Notice:

The MTT solution needs to be prepared immediately before use to avoid repeated freezing and thawing.

Sterile-filtered MTT solution is recommended (to prevent microbial degradation).

Stage 2    Cell preparation and handling

1. Prepare cell samples.

Adherent cells: Digest the cells in the logarithmic growth phase, adjust the cell concentration to 5×10³-1×104 cells/well, and inoculate them in a 96-well plate with 100 µL per well.

Suspension cells: adjust the cell concentration to 1×10⁶ /mL and add 100 µL of cell suspension (containing 1×104 cells) to each well.

Note: When culturing cells, avoid using culture media containing vitamin C or phenol red (which compete with the MTT reaction).

2. According to the experimental requirements, add different concentrations of drugs to the culture medium, and reserve blank wells (culture medium only), control wells and treatment groups.

3. Place the 96-well plate in a 37℃, 5% CO2 incubator and incubate for 16-48 hours.

Stage 3    MTT staining

The presence of serum or phenol red in the culture medium can create background. If your samples contain serum or phenol red, set up a sample background control: 50 µL MTT reagent + 50 µL cell culture medium (no cells).

Prepare parallel wells as solvent controls and use the same volume of solvent as used to treat the cells.

1. Remove the culture medium from the cell culture.

For adherent cells, carefully aspirate the culture medium.

For suspension cells, centrifuge the 96-well plate at 1,000 × g in a microplate-compatible centrifuge at 4°C. Centrifuge at 500 g for 5 min and carefully aspirate the culture medium.

2. Add 50 µL serum-free medium and 50 µL MTT solution to each well and mix gently.

3. Place the well plate in a 37°C incubator and incubate for 3-4 hours until obvious blue-purple crystals appear.

4. After incubation, add 150 µL of MTT solvent (4 mM HCl, 0.1% NP40 in isopropanol) to each well.

Cover the dish with foil and place on an orbital shaker for 15 minutes.

6. Use a microplate reader to read OD=590 The absorbance of each well at nm is measured. The culture medium background is subtracted from the measured reading. This is the corrected absorbance. The amount of absorbance is proportional to the number of cells. The corrected absorbance can be used to calculate cell proliferation rate or cytotoxicity.

Note: Try to read the plate within 1 hour.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/