What is the Difference Between MTT and MTS Assay

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. They help to reveal the effect of the test molecules on cell proliferation and cytotoxicity, affecting the cell viability. Furthermore, MTT assay is more time consuming than MTS assay.

Key Areas Covered

1. What is MTT Assay
     – Definition, Process, Importance
2. What is MTS Assay
     – Definition, Process, Importance
3. What are the Similarities Between MTT and MTS Assay
     – Outline of Common Features
4. What is the Difference Between MTT and MTS Assay
     – Comparison of Key Differences

Key Terms

Cell Proliferation, Cell Viability, Cytotoxicity, MTS Assay, MTT Assay

What is MTT Assay

MTT assay is the first homogeneous cell viability assay developed for a 96-well format suitable for high throughput screening (HTS). The principle of this assay is to determine the cell viability based on the ability of cells to reduce a tetrazolium dye by the action of the enzyme, NAD(P)H-dependent cellular oxidoreductase, which is present in the viable cells. Here, the tetrazolium dye used for the MTT assay is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, which is yellow in color. The above mentioned enzymatic action results in the formation of an insoluble formazan product in the form of crystals precipitated inside cells and near the cell surface and in the culture medium. However, as the quantity of formazan product is measured by means of measuring the absorbance, the insoluble crystals have to be solubilized first and then the absorbance can be measured at 570 nm.

Figure 1: MTT Reaction

However, although the MTT assay uses fewer materials and steps, there are some disadvantages to this method. The assay is not suitable for suspending cells. Moreover, precipitated proteins and cell debris present in the sample can interfere with the absorbance measurement, reducing the sensitivity as well as the accuracy of the assay. Also, the assay time and the number of cells present in the sample have to be optimized for each cell type.

What is MTS Assay

MTS assay is a novel method for MTT assay as it uses a new type of tetrazolium dye, which ultimately results in an aqueous, soluble formazan product. Here, the tetrazolium dye used in the MTS assay is 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium. The formation of the soluble formazan dye occurs in the presence of phenazine methosulfate (PMS), which serves as an intermediate electron acceptor, transferring electrons from NADH to reduce the tetrazolium dye, forming the soluble formazan product.

Figure 2: MTT Plate

The direct formation of a soluble formazan product reduces the formazan product solubilization step of the MTT assay. Therefore, MTS assay is more efficient and less time-consuming. Also, it removes potential errors such as cell loss, which can happen during removal of the medium and solubilizing the formazan product. Additionally, the resultant formazan product is darker in color; this increases the sensitivity and the accuracy of the assay.

Similarities Between MTT and MTS Assay

• MTT and MTS assay are two types of assays that determine cell viability in vitro.
• Both assays help to assess the effect of test molecules on cell proliferation and cytotoxicity.
• Also, both are colorimetric assays.
• Furthermore, they assess the metabolic activity of cells based on the capability of cells to reduce the tetrazolium dye to its formazan.
• In addition, the enzyme responsible for the above reduction is NAD(P)H-dependent cellular oxidoreductase, which is present in viable cells.
• Moreover, the reduction reaction occurs outside the cell by plasma membrane electron transport.
• The MTT reagent is light-sensitive; hence, these assays have to be performed in dark.
• Besides, the incubation time for both assays is the same after the addition of tetrazolium dye and it is 1 to 4 hours at 37°C.

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. Thus, this is the main difference between MTT and MTS assay.

Type of Tetrazolium Dye Used in the Assay

Furthermore, while MTT assay uses MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), MTS assay uses MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium).

The solubility of the Formazan Product

Also, the formazan product in the MTT assay is insoluble while the formazan product in MTS assay is soluble. Hence, this is an important difference between MTT and MTS assay.

Measurement of Absorption

Moreover, the absorbance is measured at 570 nm in MTT assay while the absorbance is measured at 490 nm.

Time

Time is also a difference between MTT and MTS assay. MTS assay is less time consumable than MTT assay.

Sensitivity and Accuracy

Besides, MTT assay is less sensitive and less accurate as cell debris and precipitated proteins can interfere with the absorbance measurement while MTS assay is more sensitive and accurate due to the formation of the darker formazan product.

Cost

Another difference between MTT and MTS assay is the cost. MTT assay is less expensive than MTS assay.

Conclusion

MTT assay is a classical assay for measuring the cell viability to assess the effect of test molecules on cell proliferation and cytotoxicity. However, the resultant formazan product in the MTT assay is insoluble; hence, this assay requires an additional step to solubilize the formazan product. In contrast, MTS assay is a novel method for measuring cell viability and it uses a new kind of tetrazolium salt, which produces a soluble formazan, reducing one step in the MTT assay procedure. Therefore, MTS assay is more efficient when compared to the MTT assay. Hence, the main difference between MTT and MTS assay is the characteristics of the formazan product.

Reference:

1. Riss TL, Moravec RA, Niles AL, et al. Cell Viability Assays. 2013 May 1 [Updated 2016 Jul 1]. In: Sittampalam GS, Coussens NP, Brimacombe K, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-.Available Here.

Image Courtesy:

1. “MTT reaction” By Rogan Grant – Own work (CC BY-SA 4.0) via Commons Wikimedia   
2. “MTT Plate” By Shinryuu – Own work (Public Domain) via Commons Wikimedia