**3.2 WST-1 assay**

This method is used to detect the cytotoxicity of compound towards various cancer cell lines. The mitochondrial dehydrogenase enzyme cleaves the tetrazolium salt to formazan. The amount of the dye generated by the activity of

**107**

*Cell-Based Assays in Cancer Research*

cells [20].

**3.3 MTS assay**

at 490–500 nm.

**4. Viability assay in normal cells**

lymphocyte pellet in 500 μl PBS.

**4.2 Lymphocyte viability assay**

can be harvested, counted, and seeded (5 × 103

**4.1 Isolation of lymphocytes from whole blood**

*DOI: http://dx.doi.org/10.5772/intechopen.90226*

dehydrogenase is directly proportional to the number of living cells. In the WST-1 assay protocol, add the WST-1 assay reagent to the cell culture media, and incubate for between 0.5 and 4 h, shake well. The formazan crystals produced by viable cells can be quantified and can be read in microplate reader at 440 nm. Cellular proliferation represents the ability of healthy cells to divide and create progeny [19]. Therefore, cell viability assays and cell proliferation assays are used to calculate the number of healthy cells in a population or the rate of growth of a population of

MTS is a colorimetric method used to quantify viable cells in proliferation assay.

Attempts are pursued to develop drugs that are nontoxic to normal cells; mean-

In 1968, Boyum [21] described methods for the isolation of mononuclear cells from circulating blood and bone marrow. The solution contains Ficoll and sodium diatrizoate, adjusted to a density of 1.077 ± 0.001. This medium facilitates rapid recovery of viable lymphocytes from small volumes of blood on centrifugation. It is usually employed as the initial isolation step prior to enumeration of T, B, and null lymphocytes. In brief, 3 ml of blood will be taken in heparinized test tube. About 5 ml of PBS will be added and mixed well by inversion. About 3 ml of Ficoll Hypaque solution was added in a conical centrifuge tube. Carefully layered 8 ml of blood-PBS mixture on to the Ficoll Hypaque, keeping the tube in a slanting position. Centrifuged at 2000 rpm for 30 min. The opaque interface containing mononuclear cells was aspirated and transferred into a clean conical centrifuge tube with a Pasteur pipette and discard the upper layer. About 10 ml of PBS solution was added and mixed by inversion. Centrifuged at 1500 rpm for 10 min and the supernatant was discarded. Resuspend the pellet with 5 ml PBS and centrifuge at 1500 rpm for 10 min. Repeat the step thrice and resuspend the

In vitro experiments with compounds can be tested in lymphocyte using lymphocyte viability assay. The lymphocytes were aspirated from the gradient plasma interfaces and washed twice in PBS, and then the final cell pellets will be resuspended in RPMI-1640 medium containing 10% FCS and 100 μl streptomycin, and 100 μl fungicide can be added to avoid contamination; pH 7.4 is ideal. Cells

titer plates (Axygen). PBS will be added to the outer wells (200 μl/well). After 24 h of incubation at 37°C in 5% CO2 to allow cell attachment, the media will be

cells/well in 100 μl) in 96 well

while toxic to cancer cells can be considered as good anticancer agent.

The NAD(P)H-dependent dehydrogenase enzymes in metabolically active cells reduce the MTS tetrazolium compound and form colored formazan product that is soluble in cell culture media. It can be used to test cell proliferation, cell viability, and cytotoxicity. The formazan dye can be quantified by measuring the absorbance

**Figure 2.** *MTT assay: purple color indicate the viability of cells.*

#### *Cell-Based Assays in Cancer Research DOI: http://dx.doi.org/10.5772/intechopen.90226*

dehydrogenase is directly proportional to the number of living cells. In the WST-1 assay protocol, add the WST-1 assay reagent to the cell culture media, and incubate for between 0.5 and 4 h, shake well. The formazan crystals produced by viable cells can be quantified and can be read in microplate reader at 440 nm. Cellular proliferation represents the ability of healthy cells to divide and create progeny [19]. Therefore, cell viability assays and cell proliferation assays are used to calculate the number of healthy cells in a population or the rate of growth of a population of cells [20].

#### **3.3 MTS assay**

*Cell Growth*

(5 × 103

cells. The percentage of viable cells can be determined by dividing the number of unstained cells by the total number of cells and multiplying by 100. The equation is

The assay detects the living cells, and it be used to measure cytotoxicity, proliferation, or activation [18]. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay is based on the principle that mitochondrial dehydrogenase enzyme forms viable cells to cleave the tetrazolium rings of the pale yellow MTT and forms a dark blue formazan crystal which is largely impermeable to cell membranes, thus resulting in its accumulation within healthy cells. The solubilization of the cells is by the addition of a detergent that cause in the liberation of the crystals, which are solubilized. The color can be quantified using a multi-well plate reader at 570 nm. The cells can be maintained in DMEM medium, supplement with 10% FCS. Briefly, cells in the log phase of growth can be harvested, counted, and seed

 cells/well in 100 μl) in 96 well titer plates (Axygen), and add PBS to the outer wells (200 μl/well). After 24 h of incubation at 37°C in 5% CO2 to allow cell attachment, media will be removed; cultures will be treated with various concentration of compounds diluted with medium. Cells and media are used as the negative controls. The plates are further incubated for 24, 48, and 72 h. On completion of incubation, with the extract, media can be removed without disturbing the adherent cells. In the case of suspension cells lines, the media can be removed after the plates are centrifuge at 2000 rpm for 15 min. To each well, 100 μl of 5 mg/ml stock solution of MTT dye will be added, and plates can be incubated for 2 h in the dark at 37°C in a CO2 incubator. About 100 μl of lysis buffer can be added to each well, and the plates can be incubated for 4 h in the dark in a CO2 incubator and absorbance can be read using ELISA plate reader. Three replicates are set up for each concentration. The concentration required to reduce absorbance by 50% (IC50) in comparison to control cells is determined. In MTT assay colorless well indicates the cytotoxicity

This method is used to detect the cytotoxicity of compound towards various cancer cell lines. The mitochondrial dehydrogenase enzyme cleaves the tetrazolium salt to formazan. The amount of the dye generated by the activity of

Absorbance of treated cells <sup>×</sup> <sup>100</sup> \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Absorbance of control cells (1)

as follows: % of Cytotoxicity = [No. of blue cells/Total no. of cells] × 10.

**3.1 Evaluation of cytotoxicity using MTT assay**

of KB human oral cancer cells (**Figure 2**)

*MTT assay: purple color indicate the viability of cells.*

**3.2 WST-1 assay**

1 % of Growth inhibition = 100 −

**106**

**Figure 2.**

MTS is a colorimetric method used to quantify viable cells in proliferation assay. The NAD(P)H-dependent dehydrogenase enzymes in metabolically active cells reduce the MTS tetrazolium compound and form colored formazan product that is soluble in cell culture media. It can be used to test cell proliferation, cell viability, and cytotoxicity. The formazan dye can be quantified by measuring the absorbance at 490–500 nm.
