Endah Puji Septisetyani, Ratih Asmana Ningrum, Yulaika Romadhani, Popi Hadi Wisnuwardhani, Adi Santoso


A 3-(4,-5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay  is  a method that used to measure cell viability. It is based on the conversion of MTT by succinic dehydrogenase enzyme into insoluble formazan. Dissolution of formazan by using proper solvent is the most important step of MTT assay to obtain valid and reliable data. In this study, we observed several solvents [isopropanol, dimethyl sulfoxide (DMSO), and sodium dodecyl sulphate (SDS)] to validate MTT assay by using MCF-7 cells. The observation was performed by MTT addition at concentration of 0.5µg/µL, 3-4h cells incubation at 37°C, dissolution of formed formazan crystal and absorbance measurement at 570nm. The result showed that formazan completely dissolved in DMSO and 10% SDS. The most advantage of using SDS was it avoided the removal of partially dissolved formazan. In this observation, we also found that pH was a very important factor in SDS solution that affected the reaction. The use of optimal condition on MTT assay by SDS-0.01M HCl and SDS-0.025M HCl as formazan solvents showed that IC50 of curcumin were 32.3±0.78µM and 24.08±1.72µM respectively, while WST-1 assay resulted IC50 of curcumin 80.69±5.35µM. Altogether, this study strongly indicated that SDS-0.01M HCl was the best formazan solvent for MTT assay.

Full Text:

PDF (PP.245-254)


Al-Rubeai M., Weizenbach Lloyd DR., Emery AN. 1997. A rapid method for evaluation of cell number andviabillity by flow cytometry. Cytotechnology. 24(2): 161-168.

Berg K., Zhai L., Chen M., Kharazmi A., Owen TC. 1994. The use of water soluble formazan complex to quantitate the cell number and mitochondrial function of Leishmania major promastigotes. Parasitol Res. 80:235-239.

Bernard S., Pujo-Menjovet L., Mackey, M.C. 2003. Analysis of cell kinetics using a cell division marker: mathematicalmodeling of experimental data. Biophys. J. 84(5):3414-3424.

Berridge MV., Herst PM., Tan AS. 2005. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol. An. Rev. 11:127-152.

Berridge MV., Tan AS. 1993. Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Archives Biochem. Biophys. 303:474-482.

Boyd MR. 1997. The NCI in vitro anticancer drug discovery screen, cancer drug discovery & development. Edited by Teicher, B.A., Humana press, New Jersey, USA, pp. 23-42.

Boyd MR., Paull KD. 1995. Some practical considerations and applications of the national cancer institute in vitro anticancer drug discovery screen. Drug Development Research. 34:91-109.

Denizot F., Lang R. 1986. Rapid colorimetric assay for cell growth and survival. modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Meth. 89:271.

Haustermans KM., Hofland I., Van-Poppe LH., Oyen R., Van de Voordew, et al. 1997. Cell kinetic measurement in prostate cancer. Int. J. Radiat. Oncol. Biol. Phys. 37(5):1067-70.

Itagaki H., Ohno T., Hatao M., Hattoti C., Hayasaka A., et al. 1998. Validation study on five cytotoxicity assay by JSAAE-VII. Details of the MTT assay. Altern. Animal Test. Experiment. 5.

Jiao H., Shen W., Ohe Y., Miura K., Tamura T., Saijo, N. 1992. A New 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for testing macrophage cytotoxicity to l1210 and its drug-resistant cell lines in vitro. Cancer Immunol. Immunother. 35:412-416.

Klawitter M., Quero L., Klasen J., Gloess AN., Kloppaogge B., et al. 2012. Curcuma DMSO extracts and curcumin exhibit an anti-inflammantory and anti-catabolic effect on human intervertebral disc cells, possibly by influencing TLR 2 expression and JNK activity. J. Inflam. 9:29.

Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicicy assays. J. Immunol. Methods. 65:55-63.

Ongena K., Das C., Smith JL., Gil S., Johnson G. 2010. Determingcell number duringcell cultureusing sceptercell counter. J.Vis. Exp. 45:2204.

Ramachandran C., Rodriguez S., Ramachandran R., Nair PKR., Fonseca et al. 2005. Expression profiles of apoptotic genes induced by curcumin in human breast cancer and mammary epithelial cell lines. Anticancer Res. 25:3293-3302.

Rubinstein LV., Shoemaker RH., Pauli KD., Simon RM., Tosini S., et al. 1990. Comparison of in vitro anticancer drug screening data generated with a tetrazolium assay US a protein assay against a diverse panel of human tumor cell lines. J. Natl. Cancer Inst. 82(13):1107-1112. 82(13):1113-1117.

Russel CA., Vindelov LL.. 1998. Optimization and Comparison of the MTT assay and the 3H-TdR assay for the detection of IL-2 in helper T cell precurcor assays. J. Immunol. Methods. 217:165-175.

Scudiero DA., Shoemaker RH., Paul KD., Monks A., Tierrey S., et al. 1988. Evaluation of a soluble tetrazolium/ formazan assay for cell growth and drug sensivity in culture using human an othertumor cell lines. Can. Res. 48:4827-4833.

Skehan P., Storeng R., Swdiro D., Monks A., McMohan J., Vistica D., Warren JT., Bokesh H., Kenney S., Boyd MR., 1990, New colorimetric cytotoxicity assay for anticancer drug screening. J. Natl. Cancer Inst. 82(13):1107-1112.

Tada H., Shiho O., Kuroshima, K., Koyama, M., Tsukamoto, K. 1984. An improved colorimetric assay for interleukin 2. J. Immunol. Methods. 93(2):157-165.

Trivedi AB., Kitabatake N., Doi E. 1990. Toxicity of dimethyl sulfoxide as a solvent in bioassay system with HeLa cells evaluated colorimetry with 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide. Agric. Biol. Chem. 54(11):2961-2966.

Twentyman PR., Luscombe M. 1987. A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. Br. J. Cancer. 56:279-285.

Weaver LW. 1998.Estimation of cell viability by flow cytometry. Methods in Mol. Biol. 91:77-83.

Young FM., Phungtamdet W., Sanderson BJS. 2005. Modification of MTT assay conditions to examine the cytotoxic effects of amitraz on the human lymphoblastoid cell line, WIL2NS. Toxicology in Vitro. 19(8):1051-1059.

Zaidi D., Singh N., Ahmad IZ., Sharma R., Balapure AK., 2011, Antiproliferative effects of curcumin plus centchroman in MCF-7 and MDA MB-231 Cells. Int. J. Pharm. Pharmaceutical Sci. 3(2):212-216.

Zhong H., Wang F., Fan, Q., Wang L. 2012. Curcumin inhibits metastatic progression of breast cancer cell through suppression of urokinase-type plasminogen activator by NF-kappa B signaling pathways. Mol. Biol. Rep. 39:4803-4808.

Zhou Q., Wang X., Liu X., Zhang H., Lu Y., Su S. 2011. Curcumin enhanced antiproliferative effect of mitomycin C in human breast cancer MCF-7 cells in vitro and in vivo. Acta Pharmacologica Sinica. 32:1402-1410.

DOI: http://dx.doi.org/10.14499/indonesianjpharm25iss4pp245


  • There are currently no refbacks.


Creative Commons License
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.

Indonesian J Pharm indexed by:


analytics View My Stats