Reduction of the New Tetrazolium Dye, Alamar Blue™, in Cultured Rat Hepatocytes and Liver Fractions

Abstract

Alamar Blue™ (AB) is a new tetrazolium dye substrate that has been introduced as an alternative cell viability indicator. AB is reduced by intracellular reductases to a product which is exported from cells and can be quantified by fluorescent or spectrophotometric methods. We investigated the processes by which AB was reduced in liver cytosolic, microsomal or mitochondrial fractions and in cultured rat hepatocytes. AB reduction was catalysed by all liver fractions in an NADPH-dependent and NADH-dependent manner; the cytosolic fraction catalysed the highest rate of AB reduction. All of these activities were inhibited by dicoumarol (10μM), except AB reduction catalysed by NADH in mitochondrial fractions, which was resistant to the effects of dicoumarol and the metabolic inhibitors, but sensitive to inhibition by mercury (II) chloride. In hepatocyte cultures, AB reduction was stimulated by dicoumarol (10μM), menadione (10μM), rotenone (10μM), lactate (1–10mM) and fluoride (3-10mM). Potassium cyanide, ethanol and malonate had little effect. The results from this study suggest that AB is reduced in an NADPH-quinone oxidoreductase-dependent fashion, but that superoxide may also be involved in the reduction of AB. The modulation of AB reduction by lactate means that AB reduction may be modified by alterations in intermediary metabolism which are not a reflection of cell lethality. Therefore, great care should be exercised when using AB reduction as a viability indicator.