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

A Duncan Cromarty²  Annie M Joubert¹  Alet van Tonder² 

[1] Department of Physiology, University of Pretoria, Pretoria, South Africa;Department of Pharmacology, University of Pretoria, Pretoria, South Africa

关键词: Assay interference;    Lonidamine;    2-deoxyglucose;    3-bromopyruvate;    Sulforhodamine B assay;    Resazurin reduction assay;    Neutral red uptake assay;    MTT assay;   

Others  :  1135507
DOI  :  10.1186/s13104-015-1000-8

 received in 2014-07-09, accepted in 2015-01-27,  发布年份 2015

【 摘 要 】

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.

【 授权许可】

   
2015 van Tonder et al.; licensee BioMed Central.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]Adams CP, Brantner VV: Estimating the cost of new drug development: Is it really $802 million? Health Affair 2006, 25(2):420-8.
• [2]Allen DD, Caviedes R, Càrdenas AM, Shimahara T, Segura-Aguilar J, Caviedes PA: Cell lines as in vitro models for drug screening and toxicity studies. Drug Dev Ind Pharm 2005, 31:757-68.
• [3]Mosmann T: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983, 65:55-63.
• [4]Lü L, Zhang L, Wai MSM, Yew DTW, Xu J: Exocytosis of MTT formazan could exacerbate cell injury. Toxicol in Vitro 2012, 26:636-44.
• [5]Stockert JC, Blázquez-Casto A, Cañete M, Horobin RW, Villanueva A: MTT assay for cell viability: intracellular localisation of the formazan product is in lipid droplets. Acta Histochem 2012, 114:785-96.
• [6]Hansen MB, Nielsen SE, Berg K: Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Methods 1989, 119(2):203-10.
• [7]Van Rensburg CEJ, Anderson R, Jooné G, Myer MS, O’Sullivan JF: Novel tetramethylpiperidine-substituted phenazines are potent inhibitors of P-glycoprotein activity in a multi drug resistant cancer cell line. Anti-Cancer Drug 1997, 8:708-13.
• [8]Scudiero DA, Shoemaker RH, Paull KD, Monks A, Tierney S, Nofziger TH, et al.: Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res 1988, 8(48):4827-33.
• [9]Khabar KSA, Al-Zoghaibi F, Dzimiri M, Taha M, Al-Tuwaijri A, Al-Ahdal MN: MTS Interferon Assay: A simplified cellular dehydrogenase assay for interferon activity using a water-soluble tetrazolium salt. J Interf Cytok Res 1996, 16:31-3.
• [10]Peskin AV, Winterbourn CC: A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1). Clin Chim Acta 2000, 293:157-66.
• [11]Ganapathy-Kanniappan S, Morgan RH: The pyruvic acid analogue 3-bromopyruvate interferes with the tetrazolium reagent MTS in the evaluation of cytotoxicity. Assay Drug Dev Techn 2010, 8(2):258-62.
• [12]Brugisser R, Von Daeniken K, Jundt G, Schaffner W, Tullberg-Reinert H: Interference of plant extracts, phytoestrogens and antioxidants with the MTT tetrazolium assay. Planta Med 2002, 68:445-8.
• [13]Wang X, Ge J, Wang K, Qian J, Zou Y: Evaluation of MTT Assay for Measurement of Emodin-Induced Cytotoxicity. Assay Drug Dev Technol 2006, 4(2):203-7.
• [14]Wang P, Henning SM, Heber D: Limitations of MTT and MTS-based assays for measurements of antiproliferative activity of green tea polyphenols. PLoS One 2006, 5(4):1-10.
• [15]Wang S, Yu H, Wickliffe JK: Limitation of the MTT and XTT assays for measuring cell viability due to superoxide formation induced by nano-scale TiO2. Toxicol In Vitro 2011, 25:2147-51.
• [16]Fischer J, Prosenc MH, Wolff M, Hort N, Willumeit R, Feyerabend F: Interference of magnesium corrosion with tetrazolium-based cytotoxicity assays. Acta Biomater 2010, 6:1813-23.
• [17]Vistica DT, Skehan P, Scudiero D, Monks A, Pittman A, Boyd MR: Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production. Cancer Res 1991, 51:2515-20.
• [18]Gonzalez RJ, Tarloff JB: Evaluation of hepatic subcellular fractions for alamar blue and MTT reductase activity. Toxicol In Vitro 2001, 15:257-9.
• [19]Zhang SZ, Lipsky MM, Trump BF, Hsu IC: Neutral red (NR) assay for cell viability and xenobiotic-induced cytotoxicity in primary cultures of human and rat hepatocytes. Cell Biol Toxicol 1990, 6(2):219-34.
• [20]Goegan P, Johnson G, Vincent R: Effects of serum protein and colloid on the Alamar Blue assay in cell cultures. Toxicol In Vitro 1995, 9(3):257-66.
• [21]Rubinstein LV, Shoemaker RH, Paull KD, Simon RM, Tosini S, Skehan P, et al.: Comparison of in vitro anticancer drug screening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human tumour cell lines. J Natl Cancer Inst 1990, 82:1113-8.
• [22]Vichai V, Kirtikara K: Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc 2006, 1:1112-6.
• [23]National Cancer Institute Screening Services. NCI-60 DTP Human Tumor Cell Line Screen. http://www.cancer.gov (2014). Accessed 5 January 2015.
• [24]Gatenby RA, Gillies RJ: Glycolysis in cancer: a potential target for therapy. Int J Biochem Cell Biol 2007, 39:1358-66.
• [25]Rosbe KW, Brann TW, Holden SA, Teicher BA, Frei E III: Effect of lonidamine on the cytotoxicity of four alkylating agents in vitro. Cancer Chemother Pharmacol 1989, 25:32-6.
• [26]Warburg O, Posener K, Negelein E: Uber den stoffwechsel der tumoren. Biochem Z 1924, 152:319-44.
• [27]Kim J-W, Dang CV: Cancer’s molecular sweet tooth and the Warburg effect. Cancer Res 2006, 66(18):8927-30.
• [28]Pelicano H, Martin DS, Xu R-H, Huang P: Glycolysis inhibition for anticancer treatment. Oncogene 2006, 25:4633-46.
• [29]Borenfreund E, Puerner JA: Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol Lett 1985, 24(2–3):119-24.
• [30]Fotakis G, Timbrell JA: In vitro cytotoxicity assays: Comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicol Lett 2006, 160:171-7.
• [31]Repetto G, Del Peso A, Zurita JL: Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc 2008, 3(7):1125-31.
• [32]McMillian MK, Li L, Parker JB, Patel L, Zhong Z, Gunnett JW, et al.: An improved resazurin-based cytotoxicity assay for hepatic cells. Cell Biol Toxicol 2002, 18:157-73.
• [33]Jabbar SAB, Twentyman PR, Watson JV: The MTT assay underestimates the growth inhibitory effects of interferons. Br J Cancer 1989, 60:523-8.
• [34]Hamid R, Rotshteyn Y, Rabadi L, Parik H, Bullock P: Comparison of alamar blue and MTT assays for high through-put screening. Toxicol In Vitro 2004, 18:703-10.
• [35]Al-Nasiry S, Geusens N, Hanssens M, Luyten C, Pijnenborg R: The use of Alamar Blue assay for quantitative analysis of viability, migration and invasion of choriocarcinoma cells. Hum Reprod 2007, 22(5):1304-9.
• [36]Hayon T, Dvilansky A, Shpilberg O, Nathan I: Appraisal of the MTT-based assay as useful tool for predicting drug chemosensitivity in leukaemia. Leukaemia Lymphoma 2003, 44(11):1957-62.
• [37]Sieuwert AM, Klijn JG, Peters HA, Foekens JA: The MTT tetrazolium salt assay scrutinised: how to use this assay reliably to measure metabolic activity of cell cultures in vitro for the assessment of growth characteristics, IC50 values and cell survival. Eur J Clin Chem Biochem 1995, 33:813-23.
• [38]Keepers YR, Pizao PE, Peters GJ, Van Ark-Otte J, Winograd B, Pinedo HM: Comparison of the Sulforhodamine B protein and tetrazolium (MTT) assays for in vitro chemosensitivity testing. Eur J Cancer 1991, 27(7):897-900.
• [39]Queirόs O, Preto A, Pacheco A, Pinheiro C, Azevedo-Silva J, Moreira R, et al.: Butyrate activates the monocarboxylate transporter MCT4 expression in breast cancer cells and enhances the antitumour activity of 3-bromopyruvate. J Bioenerg Biomembr 2012, 44:141-53.
• [40]Zhang F, Aft RL: Chemosensitising and cytotoxic effects of 2-deoxy-D-glucose on breast cancer cells. J Cancer Res Ther 2009, 1(5):S41-3.