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.
Tetrazolium salts are widely used to investigate cytotoxicity and cell proliferation and viability. The first assay of this type was described by Mosmann et al. in 1983 (1). These authors used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) for their experiments. MTT is a water-soluble yellow dye that is converted into a water-insoluble dark blue formazan by reductive cleavage of the tetrazolium ring. The necessary step of discarding the medium and the solubilization of the formazan prior to its quantification can be troublesome and lead to errors, as reviewed in Marshall et al. (2).
For this reason, other tetrazolium salts have been described to save time and eliminate potential errors. These are 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), sodium 4-(3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolium)-1,3-benzene disulfonate (WST-1), and sodium 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) (3,4). These reagents require an electron coupling reagent for optimal formazan yield. For this, phenazine methosulfate (PMS) is normally used (5).
Slater et al. (6) demonstrated that the mitochondrial succinate dehydrogenase is involved in the cellular reduction of tetrazolium salts, but as reviewed in Marshall et al. (2), other cellular components can contribute to the formation of the formazan. The MTT assay also failed to show growth inhibition by genistein because of changes in mitochondrial reductase activity (7) and interferons, due to changes in pH of conditioned medium (8).
Huang et al. (12) showed that serum albumin leads to a decreased signal in the MTS assay in a cell-free system containing ascorbic acid as reducing agent. However, when we used the XTT assay to investigate the effect of albumin conjugates—which we develop as targeted drugs (13,—on the viability of cells, we saw an increase in the signal in the cell-free controls.
We investigated this effect further and demonstrate here a reducing effect of serum albumin in the XTT and the MTT assay, while showing the reasons for it.
XTT, PMS, MTT [≥97.5% thin layer chromotography (TLC)], and human serum albumin (HSA; minimum 96%) were purchased from Sigma-Aldrich (Steinheim, Germany). Dulbecco's phosphate-buffered saline (DPBS w/o Mg and Ca) was from PAN Biotech GmbH (Aidenbach, Germany), and bovine serum albumin (BSA; fraction V, immunoglobulin/protease free) was from Rockland Immunochemicals (Gilbertsville, PA, USA). Cysteine [L-cysteine hydrochloride monohydrate, minimum 98% thin layer chromatography (TLC)] and N-ethylmaleimide (NEM) was purchased from Sigma-Aldrich. Pronase (from Streptomyces griseus) was from Roche Diagnostics (Mannheim, Germany). Glutathione (GSH; reduced, crystallized) was from Boehringer Mannheim (Mannheim, Germany), and HSA was from U.S. Biological (Swampscott, MA, USA). Ethanol (puriss p.a.) was from Sigma-Aldrich. Vivaspin concentrators [Vivaspin 0.5 mL concentrator 5000 molecular weight cutoff (MWCO) polyethersulfone (PES)] were from Sartorius (Goettingen, Germany), and 96-well plates (BD Falcon Microtest™ 96) from BD Biosciences (Bedford, MA, USA).
Different serum albumin preparations at increasing concentrations in DPBS were tested in 96-well plates in the XTT assay. Cysteine and GSH dissolved in DPBS at equimolar concentrations to the albumin used were tested in parallel. The total volume of the solutions was 150 µL to which 50 µL XTT (1 mg/mL in DPBS) and 1 µL PMS (0.383 mg/mL in DPBS) were added. Plates were shaken and incubated at 37°C for 4 h. After the incubation, absorbance at 480 nm was read using a microplate reader (µQuant™; Bio-Tek Instruments, Winooski, VT, USA); 680 nm was used as reference wavelength. The same concentrations of HSA in DPBS were used for the MTT assay. The volume of the samples was 100 µL. Ten microliters of MTT solution (10 mg/mL in DPBS) were added to each well and incubated for 4 h at 37°C. Each hour, the plate was shaken. After the incubation period, 100 µL isopropanol were added per well, and absorbance was read at 540 nm using the same plate reader as for the XTT assay; 690 nm was used as reference wavelength. For Pronase treatment, a 10% solution of HSA in DPBS was digested with 10 mg/mL Pronase at 40°C overnight. The undigested protein was separated from peptides and amino acids by Vivaspin concentrators with a cutoff of 5000 Da. These were centrifuged at 15,000× g for 30 min. One hundred fifty microliters of the filtrate were used for the XTT assay, which was performed as previously described. To check the role of sulfhydryl (SH) groups in the observed reductive activity, these were inactivated by treating with NEM. NEM was prepared as a 50-mg/mL (400 mM) stock solution in ethanol and was diluted to 13 mM in a 10% solution of HSA in DPBS or an equimolar concentration of GSH or cysteine (1.5 mM). As a control, the same amount of ethanol was used. For NEM to react with cysteine residues, the samples were incubated at 37°C for 1 h. Afterward, 50 µL XTT reagent were added, and the XTT assay was performed. A control with DPBS, NEM, and XTT was included.