Tetrazolium dyes as tools in cell biology: New insights into their cellular reduction

Abstract

Tetrazolium salts have become some of the most widely used tools in cell biology for measuring the metabolic activity of cells ranging from mammalian to microbial origin. With mammalian cells, fractionation studies indicate that the reduced pyridine nucleotide cofactor, NADH, is responsible for most MTT reduction and this is supported by studies with whole cells. MTT reduction is associated not only with mitochondria, but also with the cytoplasm and with non-mitochondrial membranes including the endosome/lysosome compartment and the plasma membrane. The net positive charge on tetrazolium salts like MTT and NBT appears to be the predominant factor involved in their cellular uptake via the plasma membrane potential. However, second generation tetrazolium dyes that form water-soluble formazans and require an intermediate electron acceptor for reduction (XTT, WST-1 and to some extent, MTS), are characterised by a net negative charge and are therefore largely cell-impermeable. Considerable evidence indicates that their reduction occurs at the cell surface, or at the level of the plasma membrane via trans-plasma membrane electron transport. The implications of these new findings are discussed in terms of the use of tetrazolium dyes as indicators of cell metabolism and their applications in cell biology.

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.