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Linearity Comparison of Three Colorimetric Cytotoxicity Assays

DOI: 10.4236/jct.2019.107047, PP. 580-590

Keywords: Linearity, Cytotoxicity, Proliferation, Metabolism, Cancer

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Abstract:

Cell viability assays, including techniques to assess the proliferation of cancer cell lines, constitute a rapid, inexpensive and sensitive screening method to pre-clinically evaluate the activity of a potential drug or substance. This study investigates and compares seeding densities and linearity of three such methods: MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), SRB (sulforhodamine B) and CVE (crystal violet elution) assays. SRB and CVE are staining assays for proteins, while MTT measures the mitochondrial activity of living cells. Assays were performed on five cancer cell lines, A375, PC3, DU145, HCT116 and COR-L105, and the coefficient of determination (R2) was employed to determine fit into a linear regression model. The results show that CVE is the most linear assay at fixed time points. SRB at 515 nm is better for measurements over time. Seeding densities between 9000 and 12,000 were the optimum. However, seeding densities and doubling times should be taken into consideration when designing an experiment.

References

[1]  Posimo, J.M., Unnithan, A.S., Gleixner, A.M., Choi, H.J., Jiang, Y., Pulugulla, S.H. and Leak, R.K. (2014) Viability Assays for Cells in Culture. Journal of Visualized Experiments, 83, e50645.
https://doi.org/10.3791/50645
[2]  Berridge, M.V. and Tan, A.S. (1993) Characterization of the Cellular Reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT): Subcellular Localization, Substrate Dependence, and Involvement of Mitochondrial Electron Transport in MTT Reduction. Arch BiochemBiophys, 303, 474-482.
https://doi.org/10.1006/abbi.1993.1311
[3]  Mosmann, T. (1983) Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays. Journal of Immunological Methods, 65, 55-63.
https://doi.org/10.1016/0022-1759(83)90303-4
[4]  Vega-Avila, E. and Pugsley, M.K. (2011) An Overview of Colorimetric Assay Methods Used to Assess Survival or Proliferation of Mammalian Cells. Proceedings of the Western Pharmacology Society, 54, 10-14.
[5]  Feoktistova, M., Geserick, P. and Leverkus, M. (2016) Crystal Violet Assay for Determining Viability of Cultured Cells. Cold Spring Harbor Protocols, 2016, pdb.prot087379.
https://doi.org/10.1101/pdb.prot087379
[6]  Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, J.T., Bokesch, H., Kenney, S. and Boyd, M.R. (1990) New Colorimetric Cytotoxicity Assay for Anti-Cancer-Drug Screening. Journal of the National Cancer Institute, 82, 1107-1112.
https://doi.org/10.1093/jnci/82.13.1107
[7]  McCaffrey, T.A., Agarwal, L.A. and Weksler, B.B. (1988) A Rapid Fluorometric DNA Assay for the Measurement of Cell Density and Proliferation in Vitro. In Vitro Cellular & Developmental Biology, 24, 247-252.
https://doi.org/10.1007/BF02623555
[8]  Touhey, S. and Heenan, M. (2003) Cell Culture. In: Davey, J. and Lord, M., Eds., Essential Cell Biology, Vol. 1 Cell Structure, a Practical Approach, Oxford University Press, Oxford, 28-31.
[9]  Vichai, V. and Kirtikara, K. (2006) Sulforhodamine B Colorimetric Assay for Cytotoxicity Screening. Nature Protocols, 1, 1112-1116.
https://doi.org/10.1038/nprot.2006.179
[10]  Seo, J.A., Kim, B., Dhanasekaran, D.N., Tsang, B.K. and Song, Y.S. (2016) Curcumin Induces Apoptosis by Inhibiting Sarco/Endoplasmic Reticulum Ca2+ ATPase Activity in Ovarian Cancer Cells. Cancer Letters, 371, 30-37.
https://doi.org/10.1016/j.canlet.2015.11.021
[11]  Johnson-Ajinwo, O.R., Richardson, A. and Li, W.W. (2015) Cytotoxic Effects of Stem Bark Extracts and Pure Compounds from Margaritaria Discoidea on Human Ovarian Cancer Cell Lines. Phytomedicine, 22, 1-4.
https://doi.org/10.1016/j.phymed.2014.09.008
[12]  Martins, C.A., Leyhausen, G., Volk, J. and Geurtsen, W. (2015) Effects of Alendronate on Osteoclast Formation and Activity in Vitro. Journal of Endodontics, 41, 45-49.
https://doi.org/10.1016/j.joen.2014.07.010
[13]  Keepers, Y.P., Pizao, P.E., Peters, G.J., van Ark-Otte, J., Winograd, B. and Pinedo, H.M. (1991) Comparison of the Sulforhodamine B Protein and Tetrazolium (MTT) Assays for in Vitro Chemosensitivity Testing. European Journal of Cancer, 27, 897-900.
https://doi.org/10.1016/0277-5379(91)90142-Z
[14]  Chiba, K., Kawakami, K. and Tohyama, K. (1998) Simultaneous Evaluation of Cell Viability by Neutral Red, MTT and Crystal Violet Staining Assays of the Same Cells. Toxicology in Vitro, 12, 251-258.
https://doi.org/10.1016/S0887-2333(97)00107-0
[15]  Liu, Y., Peterson, D.A., Kimura, H. and Schubert, D. (1997) Mechanism of Cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Reduction. Journal of Neurochemistry, 69, 581-593.
https://doi.org/10.1046/j.1471-4159.1997.69020581.x
[16]  Vistica, D.T., Skehan, P., Scudiero, D., Monks, A., Pittman, A. and Boyd, M.R. (1991) Tetrazolium-Based Assays for Cellular Viability: A Critical Examination of Selected Parameters Affecting Formazan Production. Cancer Research, 51, 2515-2520.

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