Limitations of the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays

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dc.contributor.author Van Tonder, Alet
dc.contributor.author Joubert, Annie M.
dc.contributor.author Cromarty, Allan Duncan
dc.date.accessioned 2015-08-13T07:03:52Z
dc.date.available 2015-08-13T07:03:52Z
dc.date.issued 2013-02-20
dc.description Additional file 1: The IC50 concentrations of 3-bromopyruvate calculated for MCF-7 cells after 24 or 72 hour incubation calculated per experimental repeat. Results are indicated as IC50 ± SEM. Where no values are given these could not be calculated as the assay reported relative cell survival of greater than 50% at the tested concentrations (n = 4). en_ZA
dc.description Additional file 2: Interference of three glycolysis inhibitors with the NRU, RES and SRB assays in cell-free systems (n = 3). en_ZA
dc.description.abstract BACKGROUND : The tetrazolium-based MTT assay has long been regarded as the gold standard of cytotoxicity assays as it is highly sensitive and has been miniaturised for use as a high-throughput screening assay. However, various reports refer to interference by different test compounds, including the glycolysis inhibitor 3-bromopyruvate, with the conversion of the dye to coloured formazan crystals. This study assessed the linear range and reproducibility of three commonly used cell enumeration assays; the neutral red uptake (NRU), resazurin reduction (RES) and sulforhodamine B (SRB) assays, in comparison to the MTT assay. Interference between the MTT assay and three glycolysis inhibitors, 2-deoxyglucose, 3-bromopyruvate and lonidamine, was investigated. RESULTS : Data indicate that the NRU, RES and SRB assays showed the smallest variability across the linear range, while the largest variation was observed for the MTT assay. This implies that these assays would more accurately detect small changes in cell number than the MTT assay. The SRB assay provided the most reproducible results as indicated by the coefficient of determination after a limited number of experiments. The SRB assay also produced the lowest variance in the derived 50% inhibitory concentration (IC50), while IC50 concentrations of 3-bromopyruvate could not be detected using either the MTT or RES assays after 24 hours incubation. Interference in the MTT assay was observed for all three tested glycolysis inhibitors in a cell-free environment. No interferences were observed for the NRU, SRB or RES assays. CONCLUSIONS : This study demonstrated that the MTT assay was not the best assay in a number of parameters that must be considered when a cell enumeration assay is selected: the MTT assay was less accurate in detecting changes in cell number as indicated by the variation observed in the linear range, had the highest variation when the IC50 concentrations of the glycolysis inhibitors were determined, and interference between the MTT assay and all the glycolysis inhibitors tested were observed. The SRB assay performed best overall considering all of the parameters, suggesting that it is the most suitable assay for use in preclinical screening of novel therapeutic compounds with oxido-reductive potential. en_ZA
dc.description.librarian am2015 en_ZA
dc.description.sponsorship The National Research Foundation of South Africa (TTK1207072152) en_ZA
dc.description.uri http://www.biomedcentral.com/bmcpublichealth en_ZA
dc.identifier.citation Van Tonder, A, Joubert, AM & Cromarty, AD 2015, 'Limitations of the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays', BMC Research Notes, vol. 8, no. 47, pp. 1-10. en_ZA
dc.identifier.isbn 10.1186/s13104-015-1000-8
dc.identifier.issn 1756-0500
dc.identifier.uri http://hdl.handle.net/2263/49281
dc.language.iso en en_ZA
dc.publisher BioMed Central en_ZA
dc.rights © 2015 van Tonder et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. en_ZA
dc.subject MTT assay en_ZA
dc.subject 3-bromopyruvate en_ZA
dc.subject 2-deoxyglucose en_ZA
dc.subject Lonidamine en_ZA
dc.subject Assay interference en_ZA
dc.subject Sulforhodamine B (SRB) assay en_ZA
dc.subject Resazurin reduction (RES) assay en_ZA
dc.subject Neutral red uptake (NRU) assay en_ZA
dc.title Limitations of the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays en_ZA
dc.type Article en_ZA


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