Tetrazolium salts and formazan products in Cell Biology: Viability assessment, fluorescence imaging, and labeling perspectives

Abstract

For many years various tetrazolium salts and their formazan products have been employed in histochemistry and for assessing cell viability. For the latter application, the most widely used are 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and 5-cyano-2,3-di-(p-tolyl)-tetrazolium chloride (CTC) for viability assays of eukaryotic cells and bacteria, respectively. In these cases, the nicotinamide-adenine-dinucleotide (NAD(P)H) coenzyme and dehydrogenases from metabolically active cells reduce tetrazolium salts to strongly colored and lipophilic formazan products, which are then quantified by absorbance (MTT) or fluorescence (CTC). More recently, certain sulfonated tetrazolium, which give rise to water-soluble formazans, have also proved useful for cytotoxicity assays. We describe several aspects of the application of tetrazolium salts and formazans in biomedical cell biology research, mainly regarding formazan-based colorimetric assays, cellular reduction of MTT, and localization and fluorescence of the MTT formazan in lipidic cell structures. In addition, some pharmacological and labeling perspectives of these compounds are also described.

Suppressed de novo lipogenesis by plasma membrane citrate transporter inhibitor promotes apoptosis in HepG2 cells

“…The MTT assay is a colorimetric cytotoxicity assay and one of the most widely employed viability assays in biomedical research . This assay is a cost effective and rapid method for investigating cell viability or proliferation .…”

Characterization of a nontoxic pyomelanin pigment produced by the yeast Yarrowia lipolytica

“…The cytotoxicity of purified pyomelanin was determined both by the MTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide) assay and orange‐ethidium bromide staining (AO/EtBr). Mouse fibroblast NIH3T3 and human keratinocytes HaCaT were grown in 96 well plates as stated in Section Cells, media, chemicals and culture conditions .…”

Preparation of novel azidopyrazole derivatives with anticipated cytotoxic and antimicrobial activities

“…A closely similar derivative with ortho amino group, N ‐1‐((5‐azido‐3‐methyl‐1‐phenyl‐1 H ‐pyrazol‐4‐yl)methylene)benzene‐1,4‐diamine ( 18 ), showed activity against a single cancel line, Sup‐T1, with IC 50 17.76 μg/mL. The azido pyrazol thioxothiazolidin derivative, 3‐((5‐azido‐3‐methyl‐1‐phenyl‐1 H ‐pyrazol‐4‐yl)methyleneamino)‐2‐thioxothiazolidin‐4‐one ( 19d ), demonstrated activity against Molt4, Sup‐T1, and 562 cells with IC 50 13.55, 26.18, and 11.52 μg/mL, respectively (Table ) …”

“…Light absorbance values (OD = OD 570 − OD 620 ) were recorded at 570 and 620 nm using an ELISA reader (Anthoslabtec Instrument, Salzburg, Austria) for calculating the concentration that caused 50% inhibition (IC 50 ), ie, the cell concentration at which the light absorbance value of the experimental group is half that of the control group. These results were expressed as a percentage of the control ± SD established from n = 4 wells per experiment from three independent experiments …”