**3.1 MTT assay**

Tetrazolium salts are a large group of heterocyclic organic compounds that form highly colored and generally insoluble formazan upon reduction. These compounds, which were first prepared and used in 1894, have been widely used in tests for both biological redox systems and indicators of vitality. MTT is one of the first tetrazolium salts introduced by Mossman in 1983 [1, 6].

MTT molecule is a mono tetrazolium salt and its reagent is 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyl-2tl tetrazolium bromide, consisting of a positively charged quaternary tetraazide ring nucleus containing four nitrogen atoms surrounded by three aromatic rings containing two phenyl moieties and a thiazyl ring (**Figure 2**) [7, 8].

MTT reduction is one of the most commonly used methods to measure cell proliferation and cytotoxicity. It is an important substance in ongoing tests to determine cytotoxic responses to mitogens, antigenic stimuli, and growth factors. The MTT test is used not only for cell samples, but also for testing tissue cultures. It is used in skin irritation, skin corrosion, and eye irritation tests [9].

The decrease, that is, reduction, of MTT, which passes through the eukaryotic cell membrane very easily, causes the nuclear tetrazole ring to deteriorate and the formation of a violet-colored water-insoluble formazan (**Figure 3**).

As MTT is a positively charged molecule, it can easily pass through the cell and the mitochondrial inner membrane of metabolically active cells. Such viable cells reduce MTT into formazan in their mitochondria as they keep their metabolism in regular activity. The intensity of intracellular formazan produced by this redox reaction is measured via colorimetric-based system in spectrophotometry *in vitro*.

**Figure 2.** *The molecular structure of MTT tetrazolium salt.*

**Figure 3.** *Redox reaction principle of MTT assay.*

#### *Colorimetric Cytotoxicity Assays DOI: http://dx.doi.org/10.5772/intechopen.105772*

The MTT test takes place as a three-stage process. In the first step, the cells or tissue samples to be tested are exposed to the toxic substance. Tetrazolium salt is added to the sample obtained after the toxic substance is removed. In this process, MTT is used as the tetrazolium salt [8].

MTT is originally a yellow-colored substance. During cell proliferation, a reduction leads to an increase in the mitochondrial dehydrogenase enzyme activity and forms a purple crystal structure, formazan (**Figure 4**). The MTT assay is typically performed in several hours (1–4 hours) after incubation of cells with MTT. The formazan crystals that are formed after reaction are insoluble in water. In order to measure the absorbance, they must be dissolved in a suitable solvent, which solubilizes the product. Therefore, the formazan is usually dissolved with a solvent such as dimethyl sulfoxide (DSMO) or isopropanol before the measurement recording [10, 11].

The concentration of formazan dissolved in the appropriate solvent is determined by optical density at 570 nm. As a final step, the color change in the sample should be measured by the spectrophotometric method (**Figure 5**). The reason for this step is to measure cell viability. The measured OD values are considered to be a representation of the intracellular reduction of formazan concentration and thus of MTT. The cellular viability rate of the untreated control group is taken as 100% and proportional calculations are made according to this control group.

One of the reasons why the MTT assay is preferred is that it is a reliable and fast method. The fact that the MTT assay has fewer steps compared with other tests increases the reproducibility. Among the positive aspects of the MTT assays are that more than one sample can be examined with a single test mechanism and the result is sensitive [12].

As with every assay, MTT assays also have disadvantages. If these disadvantages are well understood and necessary precautions are taken, it does not show a negative effect during the application of the test.

As stated earlier, MTT formazan is insoluble in water. After the reaction, it forms needle-shaped crystal structures in the cells. Therefore, these formed crystals must be dissolved before proceeding to the measurement phase. If this dissolution step is not performed well, there may be a difference in absorbance between the wells [13].

**Figure 4.** *The illustration of the mechanism of MTT.*

**Figure 5.** *General steps of MTT assay.*

The DSMO substance used during the dissolution process of MTT formazans can have a toxic effect. Care should also be taken when adding DSMO to the wells. The pipette used during the application may damage the formazan crystals. This can create undesirable deviations in the results.

MTT formazan can be a toxic substance due to its structure. Therefore, a control group should be established for cell death observed due to MTT formazan toxicity. In this way, false-negative or -positive results can be avoided.

#### **3.2 XTT assay**

After the MTT test developed by Mossman (1983), other tetrazolium compounds such as XTT (2,3-bis(2-methyloxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) were described by Scudiero et al. in 1988 for the colorimetric method [14]. The XTT procedure is easy to measure proliferation, so it is an acceptable solution for quantifying cells and determining their viability. XTT is a method that is used to determine how cells respond to different growth factors, foreign chemicals, drugs, etc.

XTT testing is a fast, responsive, simple, and safe strategy for using the determination of cytotoxicity. It also offers a high level of sensitivity and precision yet the XTT assay's performance is highly dependent on the mitochondrial dehydrogenase activity of living cells. Therefore, some factors can affect the final absorbance reading. These factors are changes in the reducing capacity of living cells resulting from enzymatic regulation, pH, cellular ion concentration, cell cycle variation, and other environmental factors [15].

Having negatively charged ions on the structure, XTT cannot penetrate the cellular and mitochondrial membrane. Therefore, it is required to incorporate electron acceptor molecules such as phenazine methyl sulfate, phenazine ethyl sulfate, etc.

#### *Colorimetric Cytotoxicity Assays DOI: http://dx.doi.org/10.5772/intechopen.105772*

Phenazine methosulfate (PMS) is an electron acceptor often used in XTT tests. The oxidized (cationic) form of PMS has a yellow color, whereas the reduced derivative shows no color. The reduced PMS is utilized in this assay as an electron carrier since it is rapidly oxidized by oxygen. PMS can also be reduced non-enzymatically by NADH and NADPH. The reduction at the cellular level is gained by PMS at the plasma membrane level and reduces XTT outside the cells, thereby increasing the water solubility of the dye and formazan [5].

As illustrated in **Figure 6**, the tetrazolium salt XTT is reduced to orange-colored formazan by metabolically active cells. The electron acceptor molecules receive electrons from the cell and initiate the redox reaction to reduce the tetrazolium compound. The orange-colored formazan is dissolvable in water, thus the color intensity can be measured with a spectrophotometer. The number of metabolically active cells is proportional to the intensity of the color detected [3].

XTT testing is a fast, responsive, easy to use, and safe method for using the determination of cytotoxicity. It also has high sensitivity and accuracy. In contrast to some other salts, the formazan dye is soluble in aqueous solutions and can directly be quantified using a scanning multiplate spectrophotometer (ELISA-based, **Figure 7**). This enables a high degree of accuracy, allows online data processing by computers, and thus allows a high number of samples to be handled quickly and conveniently (4).

#### **3.3 MTS assay**

The MTS assay, which is produced as an alternative to the MTT assay, is an MTT analog that is generally formed by adding sulfonate, methyl, or similar groups to the MTT tetrazolium salt. The structure of the MTS tetrazolium salt is (3- (4,5-dimethylthiazol -2-yl) -5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl) -2H-tetrazolium) (**Figure 8**). It is also called one-step MTT assay because it is an analog developed to facilitate the MTT assay [16].

MTS tetrazolium salt is negatively charged by nature. As this situation does not allow passage through the cell membrane, the help of intermediate electron acceptor (IEA) molecules is needed. Electron acceptor molecules such as phenazine methyl sulfate (PMS) or phenazine ethyl sulfate (PES) enter the cell, and electron acquisition occurs from the cytoplasm or plasma membrane, and the reduction reaction of the tetrazolium salt takes place. The formazan (dark pink/red color), which is produced as a product after the reduction reaction, is easily soluble in water. Unlike the MTT assay, since the formazan formed after the reduction reaction is water-soluble, there is no need for a second procedure during the test (**Figure 9**) [17–19].

#### **Figure 6.** *Conversion of XTT to formazan by mitochondrial dehydrogenase.*

#### **Figure 7.**

*Illustration of XTT assay principle.*

**Figure 8.** *Chemical structure of MTS tetrazolium salt.*

#### **Figure 9.**

*Reduction of MTS to an aqueous soluble formazan through the transfer of electrons from NADH in the cytoplasm.*

*Colorimetric Cytotoxicity Assays DOI: http://dx.doi.org/10.5772/intechopen.105772*

The test procedure performed is the same as the MTT assay. MTS salt added to the cells in the culture medium is measured by spectrophotometer at 492 nm after the determined incubation period (30 min–4 hours). MTS assay, which has taken its place among *in vitro* cytotoxicity tests due to its advantages, is sensitive, fast, and easy to apply. Although the MTS assay provides ideal properties for cytotoxicity measurements, the level of absorbance at 492 nm depends on the incubation time applied, the cell type, and the number of cells tested. Considering all these substances, it is a suitable and prone test for use in toxicological test evaluations in the right places [10, 19].

#### **3.4 WST assay**

The WST method is another colorimetric method based on the principle of tetrazolium salts that produces a water-soluble formazan product. Among the watersoluble tetrazolium salts, the most frequently used one today is WST-1 in the form of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H tetrazolium and another frequently used WST compound is WST-8 (2-(2-methoxy-4-nitrophenyl)-3- (4-nitrophenyl)-5-(2,4-disulphophenyl)-2H tetrazolium) (**Figure 10**) [20].

After the formed formazan crystals dissolve, they can be quantified quickly and easily at an absorbance value of 450 nm in a conventional microplate reader. The absorbance value measured spectrophotometrically in the WST-1 method is related to the number of viable cells (**Figure 11**). As the proliferation increases, the absorbance value increases due to the formation of formazan salt. Since proliferating cells

#### **Figure 10.** *Chemical structure of a) WST-1 tetrazolium salt and b) WST-8 tetrazolium salt.*

**Figure 11.** *Principle of WST assay.*

show more metabolic activity than non-proliferating cells, this method determines not only cell viability and cytotoxicity, but also determines cell activation and proliferation. In this method, measurements can be made even at low cell concentrations without the use of additional agents or cell washing processes. However, it is a perfect solution for the quantification of cells and the determination of their viability without using radioactive isotopes. This method is based on the principle of measuring cell proliferation concerning various growth factors and nutritional components [21].
