MTT assay
Encyclopedia
The MTT assay and the MTS assay are colorimetric assay
s for measuring the activity of enzymes that reduce MTT or close dyes (XTT, MTS, WSTs) to formazan
dyes, giving a purple color. A main application allows to assess the viability (cell counting) and the proliferation of cells
(cell culture assays
). It can also be used to determine cytotoxicity
of potential medicinal agents and toxic materials, since those agents would stimulate or inhibit cell viability and growth.
MTT (3-(4,5-Dimethylthiazol
-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole
), is reduced to purple
formazan
in living cells. A solubilization solution (usually either dimethyl sulfoxide
, an acidified ethanol solution, or a solution of the detergent
sodium dodecyl sulfate
in diluted hydrochloric acid
) is added to dissolve the insoluble purple formazan
product into a colored solution. The absorbance of this colored solution can be quantified by measuring at a certain wavelength
(usually between 500 and 600 nm) by a spectrophotometer. The absorption maximum is dependent on the solvent employed.
XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) has been proposed to replace MTT, yielding higher sensitivity and a higher dynamic range. The formed formazan dye is water soluble, avoiding a final solubilization step.
Water soluble tetrazolium salts are more recent alternatives to MTT: they were developed by introducing positive or negative charges and hydroxy groups to the phenyl ring of the tetrazolium salt, or better with sulfonate
groups added directly or indirectly to the phenyl ring.
MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), in the presence of phenazine methosulfate (PMS), produces a formazan product that has an absorbance maximum at 490-500 nm in phosphate-buffered saline.
WSTs (Water soluble Tetrazolium salts) are a series of other water soluble dyes for MTT Assays, developed to give different absorption spectra of the formed formazans. WST-1 and in particular WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium), are advantageous over MTT in that they are reduced outside cells, combined with PMS electron mediator, and yield a water-soluble formazan. Finally, WST assays (1) can be read directly (unlike MTT that needs a solubilization step), (2) give a more effective signal than MTT, and (3) decrease toxicity to cells (unlike cell-permeable MTT, and its insoluble formazan that accumulate inside cells).
take place only when reductase enzyme
s are active, and therefore conversion is often used as a measure of viable (living) cells. However, it is important to keep in mind that other viability tests (such as the CASY cell counting technology
) sometimes give completely different results, as many different conditions can increase or decrease metabolic activity. Changes in metabolic activity can give large changes in MTT or MTS results while the number of viable cells is constant. When the amount of purple formazan produced by cells treated with an agent is compared with the amount of formazan produced by untreated control
cells, the effectiveness of the agent in causing death, or changing metabolism of cells, can be deduced through the production of a dose-response curve.
Colorimetry (chemical method)
In physical and analytical chemistry, colorimetry or colourimetry is a technique "used to determine the concentration of colored compounds in solution."...
s for measuring the activity of enzymes that reduce MTT or close dyes (XTT, MTS, WSTs) to formazan
Formazan
Formazan dyes are artificial chromogenic products of the reduction of tetrazolium salts by dehydrogenases and reductases. They have a variety of colors from dark blue to deep red to orange, depending on the original tetrazolium salt used as the substrate for the reaction.Leading examples of...
dyes, giving a purple color. A main application allows to assess the viability (cell counting) and the proliferation of cells
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
(cell culture assays
Cell culture assays
In Biomaterials Testing, a cell culture assay is any method which is used to assess the cytotoxicity of a material. This refers to the in vitro assessment of material to determine whether it releases toxic chemicals in sufficient quantities to kill cells either directly or indirectly through the...
). It can also be used to determine cytotoxicity
Cytotoxicity
Cytotoxicity is the quality of being toxic to cells. Examples of toxic agents are a chemical substance, an immune cell or some types of venom .-Cell physiology:...
of potential medicinal agents and toxic materials, since those agents would stimulate or inhibit cell viability and growth.
MTT and related tetrazolium salts
Thiazole
Thiazole, or 1,3-thiazole, is a heterocyclic compound that contains both sulfur and nitrogen; the term 'thiazole' also refers to a large family of derivatives. Thiazole itself is a pale yellow liquid with a pyridine-like odor and the molecular formula C3H3NS...
-2-yl)-2,5-diphenyl
Tetrazole
Tetrazoles are a class of synthetic organic heterocyclic compound, consisting of a 5-member ring of four nitrogen and one carbon atom . The simplest is tetrazole itself, CN4H2. They are unknown in nature...
), is reduced to purple
Purple
Purple is a range of hues of color occurring between red and blue, and is classified as a secondary color as the colors are required to create the shade....
formazan
Formazan
Formazan dyes are artificial chromogenic products of the reduction of tetrazolium salts by dehydrogenases and reductases. They have a variety of colors from dark blue to deep red to orange, depending on the original tetrazolium salt used as the substrate for the reaction.Leading examples of...
in living cells. A solubilization solution (usually either dimethyl sulfoxide
Dimethyl sulfoxide
Dimethyl sulfoxide is an organosulfur compound with the formula 2SO. This colorless liquid is an important polar aprotic solvent that dissolves both polar and nonpolar compounds and is miscible in a wide range of organic solvents as well as water...
, an acidified ethanol solution, or a solution of the detergent
Detergent
A detergent is a surfactant or a mixture of surfactants with "cleaning properties in dilute solutions." In common usage, "detergent" refers to alkylbenzenesulfonates, a family of compounds that are similar to soap but are less affected by hard water...
sodium dodecyl sulfate
Sodium dodecyl sulfate
Sodium dodecyl sulfate , sodium laurilsulfate or sodium lauryl sulfate is an organic compound with the formula CH311OSO3Na). It is an anionic surfactant used in many cleaning and hygiene products...
in diluted hydrochloric acid
Hydrochloric acid
Hydrochloric acid is a solution of hydrogen chloride in water, that is a highly corrosive, strong mineral acid with many industrial uses. It is found naturally in gastric acid....
) is added to dissolve the insoluble purple formazan
Formazan
Formazan dyes are artificial chromogenic products of the reduction of tetrazolium salts by dehydrogenases and reductases. They have a variety of colors from dark blue to deep red to orange, depending on the original tetrazolium salt used as the substrate for the reaction.Leading examples of...
product into a colored solution. The absorbance of this colored solution can be quantified by measuring at a certain wavelength
Wavelength
In physics, the wavelength of a sinusoidal wave is the spatial period of the wave—the distance over which the wave's shape repeats.It is usually determined by considering the distance between consecutive corresponding points of the same phase, such as crests, troughs, or zero crossings, and is a...
(usually between 500 and 600 nm) by a spectrophotometer. The absorption maximum is dependent on the solvent employed.
XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) has been proposed to replace MTT, yielding higher sensitivity and a higher dynamic range. The formed formazan dye is water soluble, avoiding a final solubilization step.
Water soluble tetrazolium salts are more recent alternatives to MTT: they were developed by introducing positive or negative charges and hydroxy groups to the phenyl ring of the tetrazolium salt, or better with sulfonate
Sulfonate
A sulfonate is a salt or ester of a sulfonic acid. It contains the functional group R-SO2O-.- Sulfonate salts:Anions with the general formula RSO2O− are called sulfonates. They are the conjugate bases of sulfonic acids with formula RSO2OH. As sulfonic acids tend to be strong acids, the...
groups added directly or indirectly to the phenyl ring.
MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), in the presence of phenazine methosulfate (PMS), produces a formazan product that has an absorbance maximum at 490-500 nm in phosphate-buffered saline.
WSTs (Water soluble Tetrazolium salts) are a series of other water soluble dyes for MTT Assays, developed to give different absorption spectra of the formed formazans. WST-1 and in particular WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium), are advantageous over MTT in that they are reduced outside cells, combined with PMS electron mediator, and yield a water-soluble formazan. Finally, WST assays (1) can be read directly (unlike MTT that needs a solubilization step), (2) give a more effective signal than MTT, and (3) decrease toxicity to cells (unlike cell-permeable MTT, and its insoluble formazan that accumulate inside cells).
MTT Assays significance
These reductionsRedox
Redox reactions describe all chemical reactions in which atoms have their oxidation state changed....
take place only when reductase enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
s are active, and therefore conversion is often used as a measure of viable (living) cells. However, it is important to keep in mind that other viability tests (such as the CASY cell counting technology
CASY cell counting technology
CASY technology is an electric field multi-channel cell counting system. It was first marketed by Schärfe System GmbH in 1998. In 2006, Schärfe System was acquired by Innovatis, a company focused on cell culture analysis. CASY utilizes the techniques of electric current exclusion and pulse area...
) sometimes give completely different results, as many different conditions can increase or decrease metabolic activity. Changes in metabolic activity can give large changes in MTT or MTS results while the number of viable cells is constant. When the amount of purple formazan produced by cells treated with an agent is compared with the amount of formazan produced by untreated control
Scientific control
Scientific control allows for comparisons of concepts. It is a part of the scientific method. Scientific control is often used in discussion of natural experiments. For instance, during drug testing, scientists will try to control two groups to keep them as identical and normal as possible, then...
cells, the effectiveness of the agent in causing death, or changing metabolism of cells, can be deduced through the production of a dose-response curve.