Tetrazolium dyes as tools in cell biology: New insights into their cellular reduction
Introduction
The reduction of tetrazolium salts from colourless or weakly coloured, aqueous solutions to brightly coloured derivatives known as formazans, has been the basis of their use as vital dyes in redox histochemistry and in biochemical applications for more than half a century [1, 2, 3, 4]. Whereas most histological applications have involved ditetrazolium salts such as nitroblue tetrazolium (NBT) that form insoluble formazans, most cell-based applications have favoured monotetrazolium salts, the most widely used being MTT. Because MTT also forms an insoluble formazan it has usually been applied in endpoint assays. Other monotetrazolium salts such as XTT, MTS and more recently WST-1, are used in conjunction with intermediate electron acceptors (IEAs) that facilitate dye reduction. They form soluble formazans and consequently can be used in real time assays. The vast majority of cellular applications of tetrazolium dyes involve microplate assays that measure cell proliferation where it is assumed that dye reduction will be proportional to the number of viable cells in exponential growth phase. Although this is usually a good approximation for defined growth conditions with a particular cell type averaged across the cell cycle, problems often arise when growth conditions are non-ideal or when growth-modifying agents are used. In these situations, dye reduction will be dependent not only on cell type and number, but also on the site of action of the compound, the tetrazolium salt used and its subcellular site of reduction. A critical review of the use of tetrazolium assays to measure cell growth and function, a decade ago now [5], summarised thinking at that time about the mechanisms of bioreduction and discussed limitations surrounding the use of these microculture assays. It was suggested that these assays measure the integrated pyridine nucleotide redox status of cells.
This review will focus primarily on new knowledge about cellular reduction of the most commonly used monotetrazolium salts with particular emphasis on understanding their site of reduction and applications in cell biology. Although it is generally assumed that tetrazolium salt reduction is intracellular and related to energy metabolism, most reduction appears to be non-mitochondrial, and several tetrazolium salts are now known to be reduced extracellularly by electron transport across the plasma membrane. These unexpected findings prompt a re-evaluation of the way we consider and use tetrazolium dyes in cell-based applications.
Section snippets
Tetrazolium salts
The unique chemical and biological properties of tetrazolium salts that have led to their widespread application in histochemistry, cell biology, biochemistry and biotechnology depend on the positively charged quaternary tetrazole ring core containing four nitrogen atoms. This central structure is surrounded by three aromatic groups that usually involve phenyl moieties (Fig. 1). Following mild reduction, tetrazolium structures transform from colourless or weakly coloured salts into brightly
Cellular reduction of tetrazolium salts
The use of tetrazolium salts in cell biology initially favoured compounds that were both water-soluble and lipophilic, but in retrospect it is likely that the net positive charge on these molecules was the primary factor responsible for their successful application in cell biology. This net positive charge would have facilitated cellular uptake via the plasma membrane potential.
Mediators of tetrazolium dye reduction (intermediate electron acceptors)
In early histochemical applications, the intermediate electron carrier, PMS, was used in conjunction with tetrazolium salts to localise sites of NAD(P)H production [40]. 1-methoxyPMS (mPMS) was later introduced by Hisada and Yagi [41] as a photochemically stable electron mediator with greater efficiency and lower background in some applications. It is worth noting that mPMS was also favoured for extra-mitochondrial assays because it failed to penetrate the mitochondrial membrane [42]. With
Cofactor requirement for tetrazolium dye reduction
In the 1960s and 70s, tetrazolium salts were widely used to study the mitochondrial respiratory chain and, based on inhibitor studies, the main sites of NBT and MTT reduction were shown to be Complex I and Complex II respectively [4, 47]. It is not surprising therefore that cellular reduction of MTT came to be associated with the flavin-containing enzyme, succinate dehydrogenase (SDH), and that mitochondria became established as the main cellular sites of tetrazolium salt reduction. Little
Cellular sites of tetrazolium dye reduction
Many oxidoreductase enzymes are capable of catalysing electron transfer from an electron donor to an acceptor tetrazolium salt. In many cases, particularly those that do not involve superoxide, an IEA such as PMS may be required to facilitate dye reduction or to enhance the rate of reduction. Although many cofactors and metabolites are potential donors of reducing electrons, NADH, NADPH, succinate and pyruvate have been the main focus of attention. The most commonly studied systems are the
Cell proliferation and drug screening assays
The use of microplate tetrazolium assays to measure cell proliferation has increased exponentially since their introduction by Mosmann in 1983 [14]. Nevertheless, these assays do not actually measure the number of viable cells in a culture or their growth but rather, an integrated set of enzyme activities that are related in various ways to cell metabolism. They utilise the cofactor, NADH, and with MTT, other substrates like succinate and pyruvate may also contribute to their reduction.
Cell viability testing
The use of cell-permeable tetrazolium salts as vital dyes in seed testing was one of their earliest technological applications [1, 66]. In this assay the ability of imbibed seeds to take up and reduce tetrazolium dyes like TTC and NBT is measured and these methodologies are still in use in some laboratories today [67]. These early cell viability tests laid the foundation for the current wide use of tetrazolium salts in cell biology where most applications depend on uptake by viable cells and
The use of tetrazolium salts to measure superoxide production
The ability of superoxide to reduce tetrazolium salts such as NBT [71] is the basis of their application in cellular assays for measuring superoxide production and granulocytic cell function in diseases like chronic granulomatous disease [9, 12, 72]. Professional phagocytes generate large amounts of superoxide following exposure to microorganisms and chemical mediators of inflammation, and this is associated with a substantial increase in cyanide-resistant oxygen consumption. This “respiratory
Microbiological applications of tetrazolium dye reductions
Traditional microbiological enumeration techniques such as colony counts on plate employing selective media are time consuming and do not account for viable non-culturable cells found in many microbial ecosystems [86, 87].
A number of different tetrazolium dyes have been used to distinguish between dormant and metabolically active microbial cells. Most respiring microorganisms are able to reduce tetrazolium dyes in their electron transport chain, generating results within hours. For example, MTT
Summary and Conclusions
The wide use of tetrazolium dyes in cell biology belies our ignorance about their biological chemistry and the nature of their cellular reduction. With the rapidly increasing use of these dyes as convenient and inexpensive tools in cell microculture applications, and the introduction of new generation tetrazolium dyes that are reduced to soluble formazans that equilibrate rapidly in the cell culture medium, there is an urgent need to understand their bioreduction so that their use can be
Acknowledgements
We thank Rob Smith and Alfons Lawen for helpful discussions, Elizabeth Chia for drawing the chemical structures and Martijn Jasperse for help with the graphics. This work was supported by the Cancer Society of New Zealand, the Marsden Fund, and a James Cook Research Fellowship to MVB.
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