Serum albumin leads to false-positive results in the XTT and the MTT assay

Abstract

Tetrazolium salts like 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) or sodium 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) that form formazans after reduction are widely used to investigate cell viability. Besides cellular enzymes, some constituents of cell media and other substances reduce tetrazolium salts, thereby interfering with these assays. We describe here that different preparations of serum albumin from bovine or human origin can lead to a concentration-dependent increase in the signals of the XTT assay; therefore leading to an overestimation of cell numbers and to an underestimation of potential cytotoxic effects of compounds to be tested. The same effect was seen in the MTT assay with human serum albumin (HSA). We demonstrate that this reductive activity cannot be inactivated by proteolytic digestion, but that it is due to the free cysteine residue in albumin, and is also observed when cysteine or glutathione (GSH) are used. Binding of N-ethylmaleimide (NEM) to the free cysteine residue leads to a decrease of the albumin interference in the XTT assay.

Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in over/underestimation of cell viability

“…Glycolysis played an important role in MTT reduction by astroglia, whereas mitochondrial oxidative metabolism -in neurons (Takahash i et al, 2002) Growth state The growth state of cells, exponential or confluent, has moderate but significant influence on MTT reduction (Liu and Dalgleish, 2009) Serum albumin In a concentration-dependent manner, human and bovine albumins increased formazan formation via direct chemical interaction with MTT or XTT. Free SH-groups of cysteine residues were implicated (Funk et al, 2007) Cholesterol Cholesterol/methyl-β-cyclodextrin complex decreased MTT reduction, enhanced exocytosis of formazan, and promoted cell type-dependent increase in oxygen consumption. No cytotoxicity of cholesterol itself was revealed in trypan blue and propidium iodide assays.…”

Original article Influence of serum and albumin on the in vitro anandamide cytotoxicity toward C6 glioma cells assessed by the MTT cell viability assay: implications for the methodology of the MTT tests

“…Huang et al [22] reported that major contributions to tetrazolium salt reduction by common culture media such as albumin and fatty acids can obscure changes in the cell number during MTT and similar assays. Funk et al [13] found that reductive activity of human serum albumin produced concentration-dependent increase in the MTT signal, although it should be mentioned that such effect did not occur when bovine serum albumin was used.…”

Ebselen Analogues Reduce 2-chloroethyl Ethyl Sulphide Toxicity in A-431 Cells

“…One reason why MTT experiments were carried out in a culture medium lacking FBS was to minimise any interference from FBS components such as albumin (41). Even though we did not assess the effects of EB-1 on extracellular redox, Schroterova et al (42) found that MTT assays provide a reliable measure of cell viability in colon cancer cells exposed to diverse selenium-containing compounds at concentrations up to 256 μmol L -1 .…”