Optimization of some parametric values of MTT for the determination of human melanoma (SK-Mel-30) cell viability

Öz

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazoliumbromide (MTT) assay is a widely used assessment method for the determination of anticancerogenic effects of active compounds including plant secondary metabolites. Recently, some important plant active ingredients have been widely investigated for anticancerogenic properties on melanoma cancer lines which are the most lethal type of skin cancer. Although some methods including DNA assay, 3H-thymidine incorporation and flow cytometry have been recommended for counting cells in the culture, MTT is one of the most frequent method and therefore, MTT assay needs to be optimized for melanoma cell lines. In this study, the MTT analytical procedure for determination of cell viability of human melanoma cell line (SK-Mel-30) was divided into nine steps and various parameters in each step (reagent amount, incubation time, centrifugation, solvent type, waiting time before spectrophotometric analysis and spectrophotometric parameters) were optimized. Optimum amount of MTT reagent and incubation time after MTT addition were determined as 10 µL and 4 h for 96 well plate, respectively. Various solvents were evaluated for solubility effectiveness of the formed crystals and DMSO was found to be the best solvent to dissolve the crystals. Waiting time before spectrophotometric reading and Uv-vis spectrums were also evaluated. At the end of the study a flowchart, presented the best analytical conditions, was constructed. Obtained findings can be used for the determination of anticancerogenic properties of plant ingredients.

Anahtar Kelimeler

• Cell viability
• Melanoma
• MTT
• Optimization
• Validation.

Kaynakça

1. Abe, K. & Matsuki, N. 2000. Measurement of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity and lactate dehydrogenase release using MTT. Neuroscience research, 38(4): 325-329. doi: 10.1016/s0168-0102(00)00188-7
2. ACS. 2019. Cancer Facts & Figures. American Cancer Society.
3. Ali, B., Kanda Kupa, L.D., Heluany, C.S., Drewes, C.C., Vasconcelos, S.N.S., Farsky, S.H.P. & Stefani, H.A. 2017. Cytotoxic effects of a novel maleimide derivative on epithelial and tumor cells. Bioorganic Chemistry, 72: 199-207. doi: https://doi.org/10.1016/j.bioorg.2017.04.013
4. Carmichael, J., DeGraff, W.G., Gazdar, A.F., Minna, J.D. & Mitchell, J.B. 1987. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res, 47(4): 936-942.
5. Chacon, E., Acosta, D. & Lemasters, J.J. 1997. 9 - Primary Cultures of Cardiac Myocytes as In Vitro Models for Pharmacological and Toxicological Assessments, 209-223. In J. V. Castell & M. J. Gómez-Lechón (Eds.), In Vitro Methods in Pharmaceutical Research, Academic Press, San Diego, 209-223 pp.
6. Denizot, F. & Lang, R. 1986. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. Journal of Immunological Methods, 89(2): 271-277. doi: 10.1016/0022-1759(86)90368-6
7. Doustvandi, M.A., Mohammadnejad, F., Mansoori, B., Tajalli, H., Mohammadi, A., Mokhtarzadeh, A., Baghbani, E., Khaze, V., Hajiasgharzadeh, K., Moghaddam, M.M., Hamblin, M.R. & Baradaran, B. 2019. Photodynamic therapy using zinc phthalocyanine with low dose of diode laser combined with doxorubicin is a synergistic combination therapy for human SK-MEL-3 melanoma cells. Photodiagnosis and Photodynamic Therapy, 28: 88-97. doi: https://doi.org/10.1016/j.pdpdt.2019.08.027
8. EUORACHEM. 2014. The fitness for purpose of analytical methods. A laboratory guide to method validation and related topics pp.

9. Hayon, T., Dvilansky, A., Shpilberg, O. & Nathan, I. 2003. Appraisal of the MTT-based assay as a useful tool for predicting drug chemosensitivity in leukemia. Leukemia & lymphoma, 44(11): 1957-1962. doi: 10.1080/1042819031000116607
10. Jabbar, S.A., Twentyman, P.R. & Watson, J.V. 1989. The MTT assay underestimates the growth inhibitory effects of interferons. British journal of cancer, 60(4): 523-528. doi: 10.1038/bjc.1989.306
11. Jo, A., Joh, H.M., Chung, J.W. & Chung, T.H. 2020. Cell viability and measurement of reactive species in gas- and liquid-phase exposed by a microwave-excited atmospheric pressure argon plasma jet. Current Applied Physics, 20(4): 562-571. doi: https://doi.org/10.1016/j.cap.2020.02.003
12. Kuete, V., Karaosmanoğlu, O. & Sivas, H. 2017. Chapter 10 - Anticancer Activities of African Medicinal Spices and Vegetables, 271-297. In V. Kuete (Ed.), Medicinal Spices and Vegetables from Africa, Academic Press, 271-297 pp.
13. Marks, D.C., Belov, L., Davey, M.W., Davey, R.A. & Kidman, A.D. 1992. The MTT cell viability assay for cytotoxicity testing in multidrug-resistant human leukemic cells. Leukemia Research, 16(12): 1165-1173. doi: https://doi.org/10.1016/0145-2126(92)90114-M
14. Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65(1): 55-63. doi: https://doi.org/10.1016/0022-1759(83)90303-4
15. MSDS. 2013. Material Safety Data Sheet: Dimethyl Sulfoxide. ScienceLab.com.
16. MSDS. 2016. Safety Data Sheet: Dimethyl Sulfoxide (DMSO). Gaylord Chemical Company, L.L.C.
17. MSDS. 2018. Material Safety Data Sheet: Ethyl alcohol 200 Proof. ScienceLab.com.
18. Pascua-Maestro, R., Corraliza-Gomez, M., Diez-Hermano, S., Perez-Segurado, C., Ganfornina, M.D. & Sanchez, D. 2018. The MTT-formazan assay: Complementary technical approaches and in vivo validation in Drosophila larvae. Acta Histochemica, 120(3): 179-186. doi: https://doi.org/10.1016/j.acthis.2018.01.006
19. Plumb, J.A. 1999. Cell sensitivity assays : the MTT assay. Methods Mol Med, 28: 25-30. doi: 10.1385/1-59259-687-8:25 Suntravat, M., Helmke, T.J., Atphaisit, C., Cuevas, E., Lucena, S.E., Uzcátegui, N.L., Sánchez, E.E. & Rodriguez-Acosta, A. 2016. Expression, purification, and analysis of three recombinant ECD disintegrins (r-colombistatins) from P-III class snake venom metalloproteinases affecting platelet aggregation and SK-MEL-28 cell adhesion. Toxicon, 122: 43-49. doi: https://doi.org/10.1016/j.toxicon.2016.09.007
20. Tubaro, A., Florio, C., Luxich, E., Vertua, R., Della loggia, R. & Yasumoto, T. 1996. Suitability of the MTT-based cytotoxicity assay to detect okadaic acid contamination of mussels. Toxicon, 34(9): 965-974. doi: https://doi.org/10.1016/0041-0101(96)00073-6
21. Twentyman, P.R. & Luscombe, M. 1987. A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. British journal of cancer, 56(3): 279-285. doi: 10.1038/bjc.1987.190
22. van Tonder, A., Joubert, A.M. & Cromarty, A.D. 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, 8(1): 47. doi: 10.1186/s13104-015-1000-8