MTT Assay – Its Principle and Protocol

Introduction

The MTT assay, abbreviated “3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide” assay, is a colorimetric assay frequently used to evaluate cell viability and proliferation. It is commonly used to investigate the impact of different substances, medications, or experimental settings on cells health because it offers valuable data about the metabolic activities of cells. MTT assay is used to measure cell viability, proliferation, and cytotoxicity. Mosmann, T. et al. were the first to describe this non-radioactive, colorimetric test technique using MTT.

Tetrazolium salts related to MTT

MTT

In living cells, MTT, a yellow tetrazole, is converted to purple formazan. To dissolve the insoluble purple formazan result into a colored solution, a solubilization solution (typically dimethyl sulfoxide, an acidified ethanol solution, or a solution of the detergent sodium dodecyl sulphate in diluted hydrochloric acid) is added. The absorbance of this colored solution can be measured using a spectrophotometer at a specific wavelength (typically between 500 and 600 nm). The amount of light absorbed is proportional to the amount of formazan accumulated inside and on the cell surface. The deeper the purple color, and hence the higher the absorbance, the higher the formazan concentration.

XTT

MTT has been proposed to be replaced with XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide), which has stronger sensitivity and a wider dynamic range. The produced formazan dye is water-soluble, eliminating the need for a further solubilization process.

MTS

In the presence of phenazine methosulfate (PMS), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) forms a formazan product with an absorbance maximum at 490 nm. The MTS test is frequently referred to as a ‘one-step’ MTT assay since it allows for the convenience of introducing reagent directly to the cell culture without the periodic stages required in the MTT experiment. However, this convenience makes the MTS assay susceptible to colorimetric interference since the intermittent stages in the MTT assay eliminate residues of colored substances, whereas, in the one-step MTS assay, these remain in the microtitre plate. When using this test, precautions must be taken to assure accuracy, and there are compelling justifications for validating MTS results with qualitative observations under a microscope. (However, this is recommended for all colorimetric assays.)

WSTs

WSTs (water-soluble tetrazolium salts) are a class of water-soluble dyes developed for MTT assays to provide various absorption spectra of the produced formazans. WST-1 and WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) are better than MTT in that they are reduced outside cells, coupled with PMS electron mediator, and generate a water-soluble formazan. Finally, WST tests (1) can be read directly (unlike MTT, which requires a solubilization step), (2) provide a more effective signal than MTT, and (3) reduce cell toxicity (unlike cell-permeable MTT and its insoluble formazan, which accumulates inside cells).

Principle

This colorimetric assay relies on metabolically active cells converting a yellow tetrazolium salt (MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) into purple formazan crystals. The MTT is converted to formazan by the NAD(P) H-dependent oxidoreductase enzymes found in the live cells. A solubilizing solution is used to dissolve the insoluble formazan crystals, The colored solution is then measured by measuring its absorbance at 500–600 nanometers with a multi-well spectrophotometer. There are more metabolically active, viable cells in a solution that is darker.

Functions of reagents:

MTT reagent:

The MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) is a mono-tetrazolium salt made up of three aromatic rings, comprising two phenyl moieties and one thiazolyl ring, surrounding a positively charged quaternary tetrazole ring core with four nitrogen atoms. When MTT is reduced, the central tetrazole ring is broken, creating the violet-blue, water-insoluble chemical known as formazan. Due to its positive charge and lipophilic nature, the MTT reagent can pass through the mitochondrial inner membrane and cell membrane of living cells, and it is converted to formazan by metabolically active cells.

Solubilizing agent:

A solvent like Dimethyl sulfoxide (DMSO) is then used to solubilize the formed water-insoluble formazan.

Required materials:

• 1X MTT Reagent having 5 ml MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), at 5 mg/mL in phosphate-buffered saline.
• Solubilizing Solution: DMSO
• Culture medium containing 10% heat inactivated FCS (fetal calf serum) and 2 mM glutamine
• Antibiotics and antifungal like penicillin/streptomycin or gentamicin
• Sample

Protocol of MTT assay

• Incubate cells in culture media with 1 g/mL actinomycin C1 for 3 hours at 37°C and 5% CO2 at a concentration of 10⁶ cells/ml.
• In tissue culture grade, 96-well microplates with flat bottoms, seed cells at a density of 5× 10⁴ per well in 100 µl of culture medium containing 1 g/ml actinomycin C1 and varying concentrations of sample in 1 to 10^th well.
• Keep the 11th well empty as negative control and the 12th well will be used as positive control containing the cell’s monolayer.
• Incubate cell culture for 24 hours at 37 °C and 5 CO₂.
• Add 10 µl of the MTT reagent (final concentration 0.5 mg/ml) to each well following the incubation period.
• Again incubate the microplate for 4 h at 37 °C and 5% CO₂. After incubation, add 100 μl of the DMSO solution into each well.
• Give overnight incubation at 37 °C and  5% CO₂). After incubation, check the complete solubilization of the purple formazan crystals
• Measure the absorbance of the samples using a microplate (ELISA) reader at 550 and 600 nm.

Results evaluation

After taking absorbance, measure cell’s survival percentage by using following formula:

Cell survival percentage = mean OD of the sample – mean OD of the neg control   ×  100

                                              mean OD of the +ve control – mean OD of the neg control

Importance of MTT assay:

Tetrazolium dye reduction is widely thought to be dependent on NAD(P)H-dependent oxidoreductase enzymes found mostly in the cell’s cytoplasm. As a result, the reduction of MTT and other tetrazolium dyes is dependent on cellular metabolic activity caused by NAD(P)H flow. MTT is reduced very little by cells with low metabolism, such as thymocytes and splenocytes. Rapidly dividing cells, on the other hand, have a high rate of MTT decrease. It is critical to remember that test conditions can change metabolic activity and consequently tetrazolium dye reduction without compromising cell viability. Furthermore, the amount of product will be determined by the process of reduction of tetrazolium dyes, which is intracellular (MTT, MTS) vs. extracellular (WST-1).

Furthermore, evidence for spontaneous MTT reduction in lipidic cellular compartments/structures without enzymatic catalysis has been given. Despite this alternate paradigm, the MTT assay still examines a cell’s reduction potential (i.e. the availability of reducing chemicals to drive cellular energetics). As a result, the final interpretation of cell viability stays unaltered. The thickness of the scaffolds may influence the MTT assay results when evaluating the viability of cells seeded on 3D fibrous scaffolds.

Applications of MTT assay:

• Quantify cell growth and viability.
• Measure cell proliferation in response to growth factors i.e. cytokines and nutrients, apoptosis and metabolic activity in response to various stimuli.
• Measure cytotoxicity.
• It studies the cell activation.
• The MTT assay helps to evaluate the effectiveness of anticancer drugs against various cancer cell lines.

Limitations of MTT assay

• There may be inefficiencies when live cells don’t have strong metabolic activity.
• The MTT test is time consuming as it has long incubation time.

Quality control of MTT assay

• To prevent experimental artefacts and incorrect results, make sure the cell lines you’re using are authentic and appropriately identified.
• Utilise MTT powder, reagents, and cell culture media of the highest quality. Inconsistent results may be the result of using contaminated or poor-quality supplies.
• To verify the sensitivity and repeatability of the assay, use the appropriate control groups, such as cells that have not been treated (negative control) and cells that have been treated with a known cytotoxic drug (positive control).
• To prevent differences in assay results due to variations in cell density, keep cell confluence constant throughout various wells.
• For each experimental condition, run the assay in triplicates or more to account for variability and assure repeatability.
• To account for any background absorbance brought on by the test components, measure the medium’s absorbance that contains MTT but doesn’t include any cells.

Precautions for MTT assay

• To avoid contamination that can result in unreliable results, maintain sterile conditions while handling cells and setting up the test.
• For the formation of formazan crystals and the MTT reagent, maintain constant incubation times. Deviations may produce unreliable outcomes.
• During the test, cover cells and MTT-formazan crystals from direct light exposure as this can cause MTT degradation and affect absorbance measurements.
• The formazan crystals are typically dissolved in dimethyl sulfoxide (DMSO). Use caution while handling DMSO because it can introduce impurities into your experiment and, at larger doses, can be hazardous to cells.
• To prevent variation in cells’ metabolism, maintain stable cell culture conditions, such as temperature, humidity, and CO2 levels.
• Use precise pipetting techniques to evenly distribute reagent volumes to each well, reducing variability.
• To guarantee homogenous cell populations, check cell viability and count using trypan blue exclusion or other suitable techniques before beginning the test.
•  Check that cells are evenly distributed and are not aggregating, as this can produce inaccurate results.
• Inaccurate readings may result from possible interference from substances that absorb light in the same wavelength range as the formazan product.
• Use appropriate data analysis techniques to determine the vitality and proliferation of cells, such as background-corrected absorbance comparisons between treated and control cells.

References

• Ghasemi M, Turnbull T, Sebastian S, Kempson IJIjoms. 2021. The MTT assay: utility, limitations, pitfalls, and interpretation in bulk and single-cell analysis.  22(23): 12827.
• Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods. 65(1-2):55-63
• https://en.wikipedia.org/wiki/MTT_assay