The main difference between MTT and MTS assay is that MTT assay has an additional step associated with the solubilization of formazan crystals whereas MTS assay is not associated with the solubilization of formazan crystals. MTT and MTS assay are two types of assays used to measure cell viability in vitro.
What are the pitfalls of the MTT assay?
Join ResearchGate to ask questions, get input, and advance your work. Be aware of pitfalls of the MTT assay (as well as MTS, XTT, WST-1) resulting in a wrong estimation of viability. Stepanenko AA, Dmitrenko VV. Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in over/underestimation of cell viability.
What is a one-step MTT assay?
The MTS assay is often described as 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 difference between MTT and WST assays?
What is the difference between MTT and WST assays? Join ResearchGate to ask questions, get input, and advance your work. Thanks Christina. MTT can readily penetrates the cells forming an insoluble formazan whereas WST cannot penetrates the cells which requires extracellularly reduced by the plasma membrane.
Why are MTMT assays usually done in the dark?
MTT assays are usually done in the dark since the MTT reagent is sensitive to light. MTT, a yellow tetrazole, is reduced to purple formazan in living cells.
What is the difference between MTS and MTT?
MTT is a tetrazolium salt that is reduced to purple formazan crystals mainly by mitochondrial succinate dehydrogenase [16]. The MTS is an alternative to MTT and the formazan formed from MTS is water-soluble and less toxic [17].
What is the MTS assay?
The MTS assay is used to assess cell proliferation, cell viability and cytotoxicity. The MTS assay protocol is based on the reduction of the MTS tetrazolium compound by viable mammalian cells (and cells from other species) to generate a colored formazan dye that is soluble in cell culture media.
What is the major difference in point of action of MTT MTS XTT and WST?
The most commonly used compounds include: MTT, MTS, XTT, and WST-1. These compounds fall into two basic categories: 1) MTT which is positively charged and readily penetrates viable eukaryotic cells and 2) those such as MTS, XTT, and WST-1 which are negatively charged and do not readily penetrate cells.
What is the difference between MTT and XTT assay?
XTT, in particular, appears more sensitive to superoxide than MTT. The reduction rate of XTT is 1.5 times that of MTT and SOD inhibition of XTT is less effective than that of MTT, indicating that XTT is more reactive with O2- than MTT.
How do you analyze MTS assay?
Calculations involved:Subtract the absorbance of the blank wells from all the wells.Divide the absorbance of the wells which have the cells treated with the drug/inhibitor by the average of the absorbances emitted from the cells in the control wells. ... Multiply the ratio by 100 to give you the viability in %.
Why MTT assay is preferred?
The MTT assay is a sensitive and reliable indicator of the cellular metabolic activity and is preferred over the other methods measuring this end-point like the ATP and 3H-thymidine incorporation assay, the latter employing radioactivity [6,7].
Why is DMSO added 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.
Why is SDS used in MTT assay?
Some of MTT assay are using DMSO as formazan solvent (Zhong et al., 2012). In our study, the use of SDS as formazan solvent showed more stabile formazan solution compare to DMSO. The use of SDS as formazan solvent also showed more reproducible data than DMSO (data not shown).
What is the principle of MTT assay?
Principle: Rapid colorimetric assay based on the cleavage of the tetrazolium ring of MTT (3-(4,5-dimethylthazolk-2-yl)-2,5-diphenyl tetrazolium bromide) by dehydrogenases in active mitochondria of living cells as an estimate of viable cell number.
What is MTS reagent?
MTS reagent is a tetrazolium inner salt useful for determining the number of viable cells in proliferation or chemosensitivity assays. MTS is bioreduced by cells into a colored formazan product that is soluble in tissue culture medium.
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).
What is the principle of XTT assay?
The assay works on the principle that the mitochondrial enzymes of healthy cells convert a yellow XTT dye into an orange compound called formazan. To begin the assay, treat the cells with methyl beta-cyclodextrin, a cholesterol-depleting agent, and subsequently sonicate the cells at low power to induce cell damage.
What is the MTS reagent?
MTS reagent is a tetrazolium inner salt useful for determining the number of viable cells in proliferation or chemosensitivity assays. MTS is bioreduced by cells into a colored formazan product that is soluble in tissue culture medium.
What is the principle of MTT assay?
Principle: Rapid colorimetric assay based on the cleavage of the tetrazolium ring of MTT (3-(4,5-dimethylthazolk-2-yl)-2,5-diphenyl tetrazolium bromide) by dehydrogenases in active mitochondria of living cells as an estimate of viable cell number.
What is 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.
What is the ATP fluorescent assay?
ATP assays are procedures that can measure cell viability based on detection of ATP. All living cells, including bacteria, can be detected with ATP assays. Several detection methods can be used, such as colorimetric, fluorescent and bioluminescent.
Most recent answer
Be aware of pitfalls of the MTT assay (as well as MTS, XTT, WST-1) resulting in a wrong estimation of viability. Stepanenko AA, Dmitrenko VV. Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in over/underestimation of cell viability. Gene. 2015 Dec 15;574 (2):193-203. doi: 10.1016/j.gene.2015.08.009.
All Answers (4)
as you noted, I generally agree that all approaches are very similar on the biochemical level.
What is MTS in a cell culture?
MTS [3- (4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium] is a new kind of tetrazolium salts, it can be reduced by viable cells to generate formazan products that are directly soluble in cell culture medium. For these improved tetrazolium reagents, the usual protocol is simply to add them to the culture medium and read the absorption of the reduced product at an appropriate time. This approach eliminates a liquid handling step during the assay procedure, thus it obviously saves time and eliminates potential errors such as cell loss that can occur when removing culture medium and subsequently solubilizing cells.
What is MTT tetrazolium reduction?
The MTT tetrazolium reduction assay was the first homogeneous cell viability assay developed for a 96-well format that was suitable for high throughput screening (HTS).
How long does it take for MTT to work?
Fast. 2-3 hours are enough for reaction while the MTT need 4 hours. Good repeatability.
What is MTT assay?
MTT assays can be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents and toxic materials. Figure 1. The process of the MTT colorimetric assay. Figure 2.
Why is cell viability assay used?
Cell viability assays are often used to screen collections of compounds to determine if the test molecules have effects on cell proliferation or show direct cytotoxic effects that eventually lead to cell death. According to the number and type of cells used, choosing a proper assay method to decided whether the expected outcome is valid or not.
What is WST-8 reduced to?
WST-8 is reduced by dehydrogenases in cells to give an orange colored product (formazan), which is soluble in the tissue culture medium. And the amount of the formazan dye generated by dehydrogenases in cells is directly proportional to the number of living cells. Figure 4. A: Structures of WST-8 and WST-8 formazan.
What color is CCK-8?
the color of CCK-8 is closed to the medium with phenol red which may miss adding regents if not careful. The operation mistake will disturb the data result.
What is a WST?
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).
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.
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.
How to dissolve purple formazan?
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. The degree of light absorption is dependent on the degree of formazan concentration accumulated inside the cell and on the cell surface. The greater the formazan concentration, the deeper the purple colour and thus the higher the absorbance.
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.
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 ...
Can WST be read directly?
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).
What is the purpose of MTT and WST?
MTT and WST assays are both colorimetric cell proliferation assays used to measure biocompatibility, which is a common pre-clinical evaluation for experimental procedure development. They are each named for the tetrazolium salt reagent that is their core component, which are reduced to their corresponding formazan dyes in living cells.
When was MTT developed?
MTT was the first used, developed in the 1980s along with the growing popularity of 96-well microplate assays. It is still widely used as the standard method to determine cell population and health, but since the MTT compound is cytotoxic and only remains stable for a few hours, other alternative assays have been developed. One of these is the WST assay, which has much lower cytotoxicity and is stable for up to 48 hours.
