Tetrazolium Reduction Assays

Among the first cell viability assays developed for HTS was the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] tetrazolium reduction assay (Mosmann 1983) that served as a milestone for this type of study. The assay offered a non-radioactive alternative to tritiated thymi-dine incorporation into DNA as a method of measuring cell proliferation. In many cases, the MTT assay can directly substitute for the tritiated thymidine incorporation assay (Figure 6.3). The MTT tetrazolium compound is prepared in a physiologically balanced solution, added to cells in culture, and incubated for approximately 4 hr. Viable cells convert MTT into an intensely colored formazan product that can be quantitated by recording changes in absorbance at specific wavelengths.

The formazan product resulting from reduction of MTT is deposited as a precipitate both inside and outside of cells. The precipitate must be solubilized before recording 570 nm absorbance that requires addition of a second reagent to generate a uniformly colored solution within the assay well. A variety of different combinations of organic solvents and detergents were developed as reagents to solubilize the formazan product, stabilize the color, avoid evaporation, and reduce interference by phenol red often present in culture medium (Tada et al. 1986; Hanson, Nielsen, and Berg 1989; Denizot and Lang 1986).

0.50

3[H]thymidine

Us 0.10

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3[H]thymidine

Us 0.10

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FIGURE 6.3 Comparison of MTT tetrazolium and tritiated thymidine incorporation assays to measure effects of hGM-CSF on proliferation of TF-1 cells. A no-cell blank absorbance of 0.065 was subtracted from all MTT values before plotting. Similar ED50 values are shown for both assays. (Source: Modified from Promega Corporation Technical Bulletin 112. CellTiter 96® Non-Radioactive Cell Proliferation Assay)

FIGURE 6.3 Comparison of MTT tetrazolium and tritiated thymidine incorporation assays to measure effects of hGM-CSF on proliferation of TF-1 cells. A no-cell blank absorbance of 0.065 was subtracted from all MTT values before plotting. Similar ED50 values are shown for both assays. (Source: Modified from Promega Corporation Technical Bulletin 112. CellTiter 96® Non-Radioactive Cell Proliferation Assay)

Tetrazolium assay technology is widely established for use in 96-well plates as evidenced by hundreds of published articles about the technique. Although the formazans are intensely colored, spectrophotometric detection has practical limits of sensitivity. The lack of sensitivity restricts the ability to miniaturize the assay into a high density plate format and has limited its adoption for HTS. The limit of detection above background using this light absorbance method is generally about 1000 cells; however, the ability to convert tetrazolium to formazan and thus sensitivity is highly dependent on metabolic activity of the cell type measured. Incubating cells for longer periods results in an accumulation of color and increased assay sensitivity up to a point, but incubation time is limited because of the toxicities of the detection reagents that utilize energy (reducing equivalents such as NADH) from the cell to generate a signal.

A significant advance in the convenience of performing tetrazolium assays occurred with the development of reagents that form aqueous soluble formazan products when chemically reduced by viable cells. This group of tetrazolium compounds includes MTS, XTT, and WST (Cory et al. 1991; Barltrop and Owen, 1981; Paull et al. 1988; Ishiyama et al. 1993; Tominaga et al. 1999). These improved reagents eliminated the need to add a second solubilization reagent and thus eliminated a liquid handling step.

The molecular charge properties of these new tetrazolium reagents that enable the formation of water-soluble formazan products also restrict membrane permeability and entry into viable cells. To overcome this problem, cell-permeable electron transfer reagents such as phenazine methosulfate, phenazine ethosulfate, and menadione are used to facilitate shuttling of electrons from cytoplasmic reducing compounds (e.g., NADH) to the tetrazolium compound in the surrounding culture medium. Although a liquid handling step is eliminated with this newer class of tetrazolium reagents, the addition of the electron transfer reagent can be considered a disadvantage because of the increased risk of adverse effects on cell physiology.

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