About MTT Assay
- Principle of MTT Assay. In MTT assay, cells in the exponential phase of growth are exposed to a cytotoxic drug. ...
- MTT Assay Requirements. Multiwell plates (96 well), Reservoir for multichannel pipette. Universal containers or tubes 8.
- Procedure of MTT Assay. Split the cells. Determine the optimal cell count for seeding in 96 well plates. ...
What is the MTT assay based on?
The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay is based on the conversion of MTT into formazan crystals by living cells, which determines mitochondrial activity. Since for most cell populations the total mitochondrial activity is related to the number of viable cells, …
What is MTMT assay used for?
MTT assay. Tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents and toxic materials. MTT assays are usually done in the dark since the MTT reagent is sensitive to light.
What is the significance of a microtiter plate after MTT assay?
A microtiter plate after an MTT assay. Increasing amounts of cells resulted in increased purple colouring. The MTT assay is a colorimetric assay for assessing cell metabolic activity. NAD (P)H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present.
What does purple mean on MTT assay?
Increasing amounts of cells resulted in increased purple colouring. The MTT assay is a colorimetric assay for assessing cell metabolic activity. NAD (P)H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present.
What is the principle of the MTT assay for cell viability measurement?
The MTT assay is a colorimetric assay for measuring cell metabolic activity. It is based on the ability of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular oxidoreductase enzymes to reduce the tetrazolium dye MTT to its insoluble formazan, which has a purple color (Fig.
What MTT assay means?
The MTT assay is a colorimetric assay for assessing cell metabolic activity. NAD(P)H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present.
How does MTT process data?
Assay protocolDiscard media from cell cultures. ... Add 50 µL of serum-free media and 50 µL of MTT solution into each well.Incubate the plate at 37°C for 3 hours.After incubation, add 150 µL of MTT solvent into each well.Wrap plate in foil and shake on an orbital shaker for 15 minutes. ... Read absorbance at OD=590 nm.
Is MTT assay qualitative or quantitative?
quantitative cytotoxicity assayThe MTT assay is a quantitative cytotoxicity assay that uses a dye called 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (abbreviated to MTT). MTT is a yellow water soluble chemical that is cleaved by mitochondrial succinate dehydrogenase to form formazan which is violet color.
Why DMSO is used in MTT assay?
We have found that DMSO is the best solvent for dissolving the formazan product, especially where a significant amount of residual medium is left in the wells of the microtitre tray used for the assay.
What is IC50 in MTT assay?
The IC50 is defined as "the concentration of an inhibitor where the response (or binding) is reduced by half." So, if you are testing viability via an MTT assay, the dose of cytotoxic compound at which you achieve 50% viability will be the IC50.
How does MTT assay determine cell proliferation?
The MTT Reagent yields low background absorbance values in the absence of cells. For each cell type the linear relation- ship between cell number and signal produced is established, thus allowing an accurate quantification of changes in the rate of cell proliferation.
What is the full name of MTT assay?
MTT Tetrazolium Assay Concept The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was the first homogeneous cell viability assay developed for a 96-well format that was suitable for high throughput screening (HTS) (1).
How do you analyze MTT results?
The data is analyzed by plotting cell number versus absorbance, allowing quantitation of changes in cell proliferation. The rate of tetrazolium reduction is proportional to the rate of cell proliferation. Where PC is the Positive control and test is the sample being tested.
What is positive control in MTT assay?
The positive control was the cells treated with 10% DMSO. After 24 hours of incubation, MTT assay was performed to evaluate the cell viability. A solution of 50 μL MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) in phosphate-buffered saline was added to each well.
Why MTT assay is preferred?
The absorbance method of detection used by the MTT assay and other tetrazolium reduction assays (i.e., MTS, XTT, WST-1, WST-8) is generally less sensitive than fluorescent and luminescent methods for detecting viable cell number.
How do you read MTT assay results?
The data is analyzed by plotting cell number versus absorbance, allowing quantitation of changes in cell proliferation. The rate of tetrazolium reduction is proportional to the rate of cell proliferation. Where PC is the Positive control and test is the sample being tested.
What is the full name of MTT assay?
MTT Tetrazolium Assay Concept The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was the first homogeneous cell viability assay developed for a 96-well format that was suitable for high throughput screening (HTS) (1).
What is the difference between MTT and MTS assay?
Definition. MTT assay refers to a colorimetric assay for assessing cell metabolic activity while MTS assay refers to a 'one-step' MTT assay which offers the convenience of adding the reagent straight to the cell culture without the intermittent steps required in the MTT assay.
What is the MTT assay?
The MTT assay is a colorimetric assay for measuring cell metabolic activity. It is based on the ability of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular oxidoreductase enzymes to reduce the tetrazolium dye MTT to its insoluble formazan, which has a purple color (Fig. 10.1 ). This assay therefore measures cell viability in terms of reductive activity as enzymatic conversion of the tetrazolium compound to water insoluble formazan crystals by dehydrogenases occurring in the mitochondria of living cells although reducing agents and enzymes located in other organelles, such as the endoplasmic reticulum are also involved ( Lu et al., 2012; Stockert et al., 2012 ). In the MTT assay a solubilization solution (dimethyl sulfoxide or 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. MTT method is one of the most widely used methods to analyze cell proliferation and viability. MTT is taken up through endocytosis and is reduced by mitochondrial enzymes as well as endosomal/lysosomal compartments, and then it is transported to cell surfaces to form needle-like MTT formazans ( Lu et al., 2012 ). It was shown that the endocytosis of MTT did not cause obvious lesion and induce cell death, but the metabolism and exocytosis of MTT could dramatically damage cells ( Lu et al., 2012 ). MTT could activate apoptosis-related factors, such as caspase-8, caspase-3 or accelerate the leakage of cell contents after the appearance of MTT formazan crystals ( Lu et al., 2012 ). Therefore, MTT method should be carefully chosen, otherwise the cell viability would be underestimated and incomparable ( Lu et al., 2012 ). The main advantage of MTT assay is the gold standard for cytotoxicity testing while the disadvantage is that the conversion to formazan crystals depends on metabolic rate and number of mitochondria resulting in many known interferences ( Lu et al., 2012; van Tonder et al., 2015 ).
What is the MTT method?
MTT method is one of the most widely used methods to analyze cell proliferation and viability. MTT is taken up through endocytosis and is reduced by mitochondrial enzymes as well as endosomal/lysosomal compartments, and then it is transported to cell surfaces to form needle-like MTT formazans ( Lu et al., 2012 ).
What are the problems with MTT reduction?
Problems: Despite the fact this method was designed for proliferation and cytotoxicity assays, it measures the activity of a mitochondrial enzyme and the signal generated is dependent on the level of cell metabolism. 276 MTT reduction assays are often erroneously described as cell proliferation assay without the use of proper controls to confirm effects on metabolism. 277 Culture conditions may affect the metabolism and consequently the rate of MTT reduction into formazan. For example, in adherent cells metabolism may be reduced when growth is inhibited by contact in a confluence cell culture situation. On the other hand the metabolism may be increased when components of the NPs can be utilized by the cell as nutrients (Fe 3 O 4, lipids, carbohydrates, etc.). Other adverse culture conditions are an altered pH or depletion of essential nutrients such as glucose. 278
What is MTT in nanoparticles?
The MTT-assay can be used to determine the viability of nanoparticle-treated cells. 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) is a yellow dye, which is reduced by cellular enzymes to the blue product formazan. Because the transformation is only possible in viable cells, the amount of blue formazan is proportional to the number of these cells ( Mosmann, 1983 ).
How to determine interference of NP?
Improvements/tricks: Suspected chemical interference of NP should be confirmed by measuring absorbance values from control wells containing culture medium, MTT and various concentrations of the NPs without cells. A variety of formazan solubilizing methods (DMSO, SDS, organic solvents, acidified isopropanol, etc.) have been used to stabilize the color, avoid evaporation, and reduce interference by culture medium components. 290–292 It is possible to change maximum absorbance at other length in order to avoid interference using new tetrazolium compounds as substrates for the MTT assay, such as MTS 293 (absorbance maximum at 490–500 nm).
How long does MTT incubate?
The MTT substrate is added to cells in culture, usually at a final concentration of 0.2–0.5 mg/mL, and incubated for 1–4 h. Then, the cells and formazan product are solubilized with an organic solvent (eg, isopropanol, DMSO, etc.) and absorbance at 570 nm is measured using a multiwell scanning spectrophotometer. 275
What is cytotoxicity analysis?
A quality of being toxic to the cells and its evaluation is termed as cytotoxicity that could be either (1) quantitative, or (2) qualitative. The examples of quantitative cytotoxicity analyses include (i) The 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H- tetrazolium bromide (MTT) Cytotoxicity Test, and (ii) The Colony Formation Cytotoxicity Test, among others. Whereas, the examples of qualitative cytotoxicity analyses include (i) Direct Contact, (ii) The Agar Diffusion Assay, and (iii) The MEM Elution assay, among others. The pristine or its modified/engineered biocomposite materials should not be toxic in nature unless it is specifically designed for targeted applications. For example, a toxic construct for drug delivery systems that targets diseased cells (e.g. cancer cells) and destroys them. For instance, research scientists can engineer cytotoxic compounds or multi materials based constructs, if they are interested in developing a therapeutic that targets rapidly dividing cancer cells. Considering other scenarios like regenerative tissue engineering, the nontoxic requirement is the norm. Thus cytotoxic evaluation of biomaterials has evolved into a sophisticated science. This particular type of toxicology also deals with methodological procedures to evaluate how well this design criterion is met when a new biomaterial is under development. In vitro cell culture assays are of principal interest to assess the biocompatibility of pristine materials or its modified/engineered biocomposite materials. Also, such initial in vitro analyses provide an excellent way to screen materials before performing in-depth in vivo based analyses.
What is MTT assay?
The MTT assay is a colorimetric assay for assessing cell metabolic activity. NAD (P)H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT 3- (4,5- di methyl thiazol -2-yl)-2,5-di phenyl tetrazolium bromide to its insoluble formazan, which has a purple color. Other closely related tetrazolium dyes including XTT, MTS and the WSTs, are used in conjunction with the intermediate electron acceptor, 1-methoxy phenazine methosulfate (PMS). With WST-1, which is cell-impermeable, reduction occurs outside the cell via plasma membrane electron transport. However, this traditionally assumed explanation is currently contended as proof has also been found of MTT reduction to formazan in lipidic cellular structures without apparent involvement of oxidoreductases.
Which cells have low MTT?
Cells with a low metabolism such as thymocytes and splenocytes reduce very little MTT. In contrast, rapidly dividing cells exhibit high rates of MTT reduction. It is important to keep in mind that assay conditions can alter metabolic activity and thus tetrazolium dye reduction without affecting cell viability.
What is a Tetrazolium dye assay?
Tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents and toxic materials. MTT assays are usually done in the dark since the MTT reagent is sensitive to light.
Is tetrazolium dye a oxidoreductase?
Tetrazolium dye reduction is generally assumed to be dependent on NAD (P)H-dependent oxidoreductase enzymes largely in the cytoso lic compartment of the cell. Therefore, reduction of MTT and other tetrazolium dyes depends on the cellular metabolic activity due to NAD (P)H flux. Cells with a low metabolism such as thymocytes ...
What is the MTT assay?
The MTT assay is a colorimetric assay for measuring cell metabolic activity. It is based on the ability of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular oxidoreductase enzymes to reduce the tetrazolium dye MTT to its insoluble formazan, which has a purple color (Fig. 10.1 ). This assay therefore measures cell viability in terms of reductive activity as enzymatic conversion of the tetrazolium compound to water insoluble formazan crystals by dehydrogenases occurring in the mitochondria of living cells although reducing agents and enzymes located in other organelles, such as the endoplasmic reticulum are also involved ( Lu et al., 2012; Stockert et al., 2012 ). In the MTT assay a solubilization solution (dimethyl sulfoxide or 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. MTT method is one of the most widely used methods to analyze cell proliferation and viability. MTT is taken up through endocytosis and is reduced by mitochondrial enzymes as well as endosomal/lysosomal compartments, and then it is transported to cell surfaces to form needle-like MTT formazans ( Lu et al., 2012 ). It was shown that the endocytosis of MTT did not cause obvious lesion and induce cell death, but the metabolism and exocytosis of MTT could dramatically damage cells ( Lu et al., 2012 ). MTT could activate apoptosis-related factors, such as caspase-8, caspase-3 or accelerate the leakage of cell contents after the appearance of MTT formazan crystals ( Lu et al., 2012 ). Therefore, MTT method should be carefully chosen, otherwise the cell viability would be underestimated and incomparable ( Lu et al., 2012 ). The main advantage of MTT assay is the gold standard for cytotoxicity testing while the disadvantage is that the conversion to formazan crystals depends on metabolic rate and number of mitochondria resulting in many known interferences ( Lu et al., 2012; van Tonder et al., 2015 ).
What is the MTT method?
MTT method is one of the most widely used methods to analyze cell proliferation and viability. MTT is taken up through endocytosis and is reduced by mitochondrial enzymes as well as endosomal/lysosomal compartments, and then it is transported to cell surfaces to form needle-like MTT formazans ( Lu et al., 2012 ).
What is cytotoxicity analysis?
A quality of being toxic to the cells and its evaluation is termed as cytotoxicity that could be either (1) quantitative, or (2) qualitative. The examples of quantitative cytotoxicity analyses include (i) The 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H- tetrazolium bromide (MTT) Cytotoxicity Test, and (ii) The Colony Formation Cytotoxicity Test, among others. Whereas, the examples of qualitative cytotoxicity analyses include (i) Direct Contact, (ii) The Agar Diffusion Assay, and (iii) The MEM Elution assay, among others. The pristine or its modified/engineered biocomposite materials should not be toxic in nature unless it is specifically designed for targeted applications. For example, a toxic construct for drug delivery systems that targets diseased cells (e.g. cancer cells) and destroys them. For instance, research scientists can engineer cytotoxic compounds or multi materials based constructs, if they are interested in developing a therapeutic that targets rapidly dividing cancer cells. Considering other scenarios like regenerative tissue engineering, the nontoxic requirement is the norm. Thus cytotoxic evaluation of biomaterials has evolved into a sophisticated science. This particular type of toxicology also deals with methodological procedures to evaluate how well this design criterion is met when a new biomaterial is under development. In vitro cell culture assays are of principal interest to assess the biocompatibility of pristine materials or its modified/engineered biocomposite materials. Also, such initial in vitro analyses provide an excellent way to screen materials before performing in-depth in vivo based analyses.
Does MTT decrease in apoptosis?
There are several limitations, however, to the MTT assay. A decrease in the concentration of D-glucose, NADH or NADPH in the culture medium may be accompanied by a decrease in MTT-formazan production. In cells undergoing apoptosis, there may be some MTT reduction at early stages, since mitochondria remain intact.
Is MTT more sensitive than trypan blue?
The MTT system is a quantitative, more sensitive test than the trypan blue exclusion assay because there is a linear relationship between cell activity and absorbance; the growth or death rate of cells can be measured. The trypan blue test is qualitative and indicates only if a cell is alive.
Overview
The MTT assay is a colorimetric assay for assessing cell metabolic activity. NAD(P)H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT, which is chemically 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, to its insoluble formazan, which h…
MTT and related tetrazolium salts
MTT, 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. …
Significance
Tetrazolium dye reduction is generally assumed to be dependent on NAD(P)H-dependent oxidoreductase enzymes largely in the cytosolic compartment of the cell. Therefore, reduction of MTT and other tetrazolium dyes depends on the cellular metabolic activity due to NAD(P)H flux. Cells with a low metabolism such as thymocytes and splenocytes reduce very little MTT. In contrast, rapidly dividing cells exhibit high rates of MTT reduction. It is important to keep in mind …
See also
• Tetrazolium chloride
• Formazan
Further reading
• Wilson AP (2000). "Chapter 7: Cytotoxicity and viability". In Masters JR (ed.). Animal Cell Culture: A Practical Approach. Vol. 1 (3rd ed.). Oxford: Oxford University Press. ISBN 978-0-19-963796-6. LCCN 00026267. OCLC 43555390. Archived from the original on 2020-04-14. Retrieved 2016-09-24.
• Bernas T, Dobrucki J (April 2002). "Mitochondrial and nonmitochondrial reduction of MTT: interaction of MTT with TMRE, JC-1, and NAO mitochondrial fluorescent probes". …
• Wilson AP (2000). "Chapter 7: Cytotoxicity and viability". In Masters JR (ed.). Animal Cell Culture: A Practical Approach. Vol. 1 (3rd ed.). Oxford: Oxford University Press. ISBN 978-0-19-963796-6. LCCN 00026267. OCLC 43555390. Archived from the original on 2020-04-14. Retrieved 2016-09-24.
• Bernas T, Dobrucki J (April 2002). "Mitochondrial and nonmitochondrial reduction of MTT: interaction of MTT with TMRE, JC-1, and NAO mitochondrial fluorescent probes". Cytometry. 47 …
External links
• MTT assay Protocol