Cell Viability and Cytotoxicity Assays
  • OVERVIEW

Cell Viability and Cytotoxicity Assays


Cell viability and cytotoxicity assays are used for drug screening and cytotoxicity tests of chemicals. 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.


MTT Cell Viability Assay

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.


XTT Cell Viability Assay

XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) has been proposed to replace MTT, yielding higher sensitivity and a higher dynamic range. The formed formazan dye is water-soluble, avoiding a final solubilization step. Water-soluble tetrazolium salts are more recent alternatives to MTT: they were developed by introducing positive or negative charges and hydroxy groups to the phenyl ring of the tetrazolium salt, or better with sulfonate groups added directly or indirectly to the phenyl ring.


MTS Cell Viability Assay

MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), in the presence of phenazine methosulfate (PMS), produces a formazan product that has an absorbance maximum at 490 nm in phosphate-buffered saline. 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. However, this convenience makes the MTS assay susceptible to colormetric interference as the intermittent steps in the MTT assay remove traces of coloured compounds, whilst these remain in the microtitre plate in the one-step MTS assay. Precautions are needed to ensure accuracy when using this assay and there are strong arguments for confirming MTS results using qualitative observations under a microscope.


WST-1 Cell Viability Assay

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 can be read directly, give a more effective signal than MTT, and decrease toxicity to cells.

Cell Viability and Cytotoxicity Assays


Cell viability and cytotoxicity assays are used for drug screening and cytotoxicity tests of chemicals. 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.


MTT Cell Viability Assay

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.


XTT Cell Viability Assay

XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) has been proposed to replace MTT, yielding higher sensitivity and a higher dynamic range. The formed formazan dye is water-soluble, avoiding a final solubilization step. Water-soluble tetrazolium salts are more recent alternatives to MTT: they were developed by introducing positive or negative charges and hydroxy groups to the phenyl ring of the tetrazolium salt, or better with sulfonate groups added directly or indirectly to the phenyl ring.


MTS Cell Viability Assay

MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), in the presence of phenazine methosulfate (PMS), produces a formazan product that has an absorbance maximum at 490 nm in phosphate-buffered saline. 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. However, this convenience makes the MTS assay susceptible to colormetric interference as the intermittent steps in the MTT assay remove traces of coloured compounds, whilst these remain in the microtitre plate in the one-step MTS assay. Precautions are needed to ensure accuracy when using this assay and there are strong arguments for confirming MTS results using qualitative observations under a microscope.


WST-1 Cell Viability Assay

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 can be read directly, give a more effective signal than MTT, and decrease toxicity to cells.

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