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

used to measure cell viability, proliferation, and cytotoxicity

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.

In living cells, MTT, a yellow tetrazole, is converted to purple formazan.

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.

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

deeper purple color = higher absorbance = formazan concentration

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 (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.

(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)

DMSO to solubilize the formed water-insoluble formazan

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.

Culture medium containing 10% heat inactivated FCS (fetal calf serum) and 2 mM glutamine

Antibiotics and antifungal like penicillin/streptomycin or gentamicin

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.

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 10th 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.

%cell survival = (mean abs of the sample - mean abs of the neg)/(mean abs of growth - mean abs negative)*100

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

Measure cell proliferation in response to growth factors i.e. cytokines and nutrients, apoptosis and metabolic activity in response to various stimuli.

test conditions can change metabolic activity and consequently tetrazolium dye reduction without compromising cell viability

The MTT assay helps to evaluate the effectiveness of anticancer drugs against various cancer cell lines.

inefficiencies when live cells don’t have strong metabolic activity

authentic and appropriates identified cell lines only

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.

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.