Turkish Journal of Biology Volume 41 Number 6 Article 7 1-1-2017 The MTT viability assay yields strikingly false-positive viabilities although the cells are killed by some plant extracts DİDEM KARAKAŞ FERDA ARI ENGİN ULUKAYA Follow this and additional works at: https://journals.tubitak.gov.tr/biology Part of the Biology Commons Recommended Citation KARAKAŞ, DİDEM; ARI, FERDA; and ULUKAYA, ENGİN (2017) "The MTT viability assay yields strikingly false-positive viabilities although the cells are killed by some plant extracts," Turkish Journal of Biology: Vol. 41: No. 6, Article 7. https://doi.org/10.3906/biy-1703-104 Available at: https://journals.tubitak.gov.tr/biology/vol41/iss6/7 This Article is brought to you for free and open access by TÜBİTAK Academic Journals. It has been accepted for inclusion in Turkish Journal of Biology by an authorized editor of TÜBİTAK Academic Journals. For more information, please contact academic.publications@tubitak.gov.tr. Turkish Journal of Biology Turk J Biol (2017) 41: 919-925 http://journals.tubitak.gov.tr/biology/ © TÜBİTAK Research Article doi:10.3906/biy-1703-104 The MTT viability assay yields strikingly false-positive viabilities although the cells are killed by some plant extracts Didem KARAKAŞ1,*, Ferda ARI 2,*, Engin ULUKAYA1,** 1Department of Medical Biochemistry, Faculty of Medicine, İstinye University, İstanbul, Turkey 2Department of Biology, Faculty of Arts and Sciences, Uludağ University, Bursa, Turkey Received: 30.03.2017 Accepted/Published Online: 16.09.2017 Final Version: 18.12.2017 Abstract: The MTT assay is one of the often used cell viability/cytotoxicity assays. However, when the methanol extracts of plants are used to test their cytotoxic potential, interference may occur, resulting in false-positive viability results. Therefore, in this study, the reliability of the MTT assay was investigated in the case of plant use. The methanol extracts of three different plants (Hypericum adenotrichum, Salvia kronenburgii, and Pelargonium quercetorum) were tested in breast cancer cell lines (MCF-7 and MDA-MB-231) using the MTT assay and the results were compared to the ATP assay, which is a much more sensitive and reliable assay due to its interference-free feature. Additionally, decreased cell density was confirmed with phase-contrast microscopy and fluorescence staining (Hoechst 33342 dye). Although both of the viability/cytotoxicity assays are considered as metabolic assays, viabilities (in %) in the MTT assay were found to be strikingly higher when compared to the results with the ATP assay. Even in the case of total death, the MTT assay still produced artificial/false increases in viability. The morphology-based evaluation of viability/cytotoxicity by phase-contrast microscopy and Hoechst 33342 staining were greatly compatible with the ATP assay results. Overestimated (false) viabilities in the MTT assay suggests a serious interference between the MTT assay itself and the extracts used. Some ingredients of plants may have reducing activity (like the dehydrogenase activity of the cells) that converts the MTT compound into the colored formazan that is the principle of the assay. Therefore, the MTT assay may not be a suitable assay for some plant extracts, urging great caution when plants are used. Key words: MTT assay, ATP assay, interference, cytotoxicity, plant extract, breast cancer 1. Introduction numbers of experiments examining a number of variables In the past years, several methods have been developed to can be readily performed (Cole, 1986). determine cell viability in cell culture (Cook and Mitchell, On the other hand, the MTT assay has some 1989). Among these methods tetrazolium salt-based disadvantages that are dependent on the cell ability to assays are widely used in order to measure cytotoxicity or overcome cell death. One remarkable disadvantage is that cell proliferation (Mosmann, 1983; Berridge et al., 1996). damaged mitochondria may be still able to reduce MTT The principle of the MTT [3-(4,5-dimethyl-2- to formazan crystals (Mosmann, 1983; Page et al., 1988; thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay Sieuwerts et al., 1995). Loveland et al. (1992) showed is the reduction of water-soluble yellow tetrazolium salt that cells with inactivated mitochondria were also able by the dehydrogenase system of metabolically active/live to produce formazan crystals as well as cells with active cells into water-insoluble blue/magenta (MTT) formazan mitochondria. Furthermore, many nonmitochondrial crystals (Morgan et al., 1998). In this way, the concentration dehydrogenases and flavin oxidases are able to reduce of dissolved formazan crystals can be quantified using a MTT (Altman, 1976; Burdon et al., 1993). Besides, spectrophotometer and it is in direct correlation to the different conditions and some chemicals/phytochemicals number of metabolically active cells (Gabrielson et al., can also lead to changes in metabolic activity (Plumb 2002; Tunney et al., 2004; Wang et al., 2010). The MTT et al., 1989; Hsu et al., 2003). The MTT compound may assay represents a simple and rapid colorimetric assay and interact with some chemicals/phytochemicals, resulting yields quantitative data (Alley et al., 1988). This assay is in false results in viability (Hsu et al., 2003; Ulukaya et carried out entirely in 96-well microtiter plates; thus, large al., 2004; Devika and Stanely Mainzen Prince, 2008). * These authors contributed equally to this work. ** Correspondence: eulukaya@istinye.edu.tr 919 KARAKAŞ et al. / Turk J Biol More importantly, investigated agents may interfere 2.3. Cell culture and chemicals with mitochondrial dehydrogenase activity, resulting in Breast cancer cell lines MCF-7 and MDA-MB-231 were activation or inhibition of mitochondrial dehydrogenases cultured in RPMI-1640 supplemented with penicillin G and thus over/underestimation of the MTT assay results (100 U/mL), streptomycin (100 µg/mL), L-glutamine, (Jaszczyszyn and Gąsiorowski, 2008). and 10% fetal bovine serum at 37 °C in a humidified The ATP assay is also used to determine cell viability. It atmosphere containing 5% CO2. is the fastest and the most sensitive cell viability assay, and Lyophilized plant extracts were dissolved in DMSO at a it is less prone to artifacts compared to other viability assay concentration of 0.05 g/0.5 mL as a stock solution. Further methods (Riss et al., 2013). Therefore, it may be considered dilutions were made in culture medium. The ATP assay as a kind of reference assay for cell viability measurement. (adenosine 5′-triphosphate (ATP) bioluminescent somatic The ATP assay was first developed by Lundin et al. as a cell assay kit), the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- somatic cell viability assay (Lundin et al., 1986). The diphenyltetrazolium bromide) substance, and all the other principle of this method is conversion of luciferin to reagents and substances were obtained commercially oxyluciferin by the luciferase enzyme in the presence of (Sigma Aldrich, USA). Mg2+ ions and ATP. The ATP assay yields a luminescent 2.4. Experimental design signal and a linear relationship exists between the intensity of the luminescent signal and the ATP concentration, and MCF-7 and MDA-MB-231 cells were seeded at a density of 4 therefore the number of cells (Mueller et al., 2004). 1 × 10 cells per well of 96-well plates in 200 µL of culture Plant extracts contain diverse chemicals and some of medium. The untreated cells received only medium and them could reduce the activity of the MTT compound, solvent (0.1% DMSO as final concentration). All of the giving false results. If this occurs, then the MTT assay cells were treated with three different plant extracts (H. has to be expected to result in false-positive results for adenotrichum, S. kronenburgii, and P. quercetorum) in the viability, regardless of any dehydrogenase activity in the range of 1.56–100 µg/mL for a period of 48 h. cell. Therefore, in this study, we aimed to investigate how 2.5. The MTT viability assay reliable the MTT assay is in the testing of the cytotoxic The MTT viability assay was performed with slight activity of plant extracts (Hypericum adenotrichum, Salvia modifications as previously described (Mosmann, 1983). kronenburgii, and Pelargonium quercetorum) at 7 different MTT was first prepared as a stock solution of 5 mg/mL concentrations (1.56–100 µg/mL) on the MCF-7 and in phosphate-buffered saline (PBS, pH 7.2) and filtered. MDA-MB-231 breast cancer cell lines in comparison with At the end of the treatment period (48 h), 20 µL of MTT the ATP assay as a reference assay. Results of our study solution was added to each well. After incubation for 4 h at indicate that the MTT assay gave false-positive results 37 °C, 100 µL: of solubilizing buffer (10% sodium dodecyl on cell viability. Due to this finding, the MTT assay has sulfate dissolved in 0.01 N HCl) was added to each well. limited application for plant extract testing on different After overnight incubation, the 96-well plate was read by cell lines. an enzyme-linked immunosorbent assay (ELISA) reader at 570 nm for absorbance density values to determine the cell 2. Materials and methods viability. The viable cells produced a dark blue formazan 2.1. Collection and identification of plants product, whereas no such staining was formed in the dead P. quercetorum was collected from Hakkari, Turkey, in June cells. The percentage of the viable cells was calculated 2006 and identified with the aid of flora books (Davis et al., using the following formula: (%) = [100 × (sample abs)/ 1988). S. kronenburgii was collected from Van, Turkey, and (control abs)]. H. adenotrichum was collected from the Kırkpınar region 2.6. The ATP viability assay at Uludağ Mountain (Bursa, Turkey) in June 2010. These The cells were seeded, grown, and treated as for the MTT specimens were identified by Prof Dr Gürcan Güleryüz, assay (see above). The ATP assay determines the level Faculty of Science, Uludağ University, Bursa, Turkey. of cellular ATP as an indirect measure of the number 2.2. Extraction of H. adenotrichum, S. kronenburgii, and of viable cells (Andreotti et al., 1995). The experiment P. quercetorum samples was performed for luminometric measurement of cell Fifteen grams of the aboveground parts of the plant samples growth (viability) according to the standard protocol of was extracted by adding 150 mL of solvent methanol the manufacturer (ATP Bioluminescence Assay, Sigma (Merck) in a Soxhlet extractor for 24 h. The crude extracts Aldrich, USA), using a count integration time of 1 s and were concentrated using a rotary evaporator at 40 °C. The a luminometer (FL×800 Microplate Fluorescent Reader, residues were then lyophilized for 42 h and stored at –20 Bio-Tek, USA). The results are given in relative light units °C prior to the testing. (RLU). 920 KARAKAŞ et al. / Turk J Biol 2.7. Fluorescence imaging the nuclei of living cells were examined after staining with Using a fluorescent microscope, cell density was detected Hoechst 33342 dye. Based on fluorescent imaging, all on the basis of the nuclear morphology. Hoechst 33342 plant extracts caused a significant decrease in cell viability dye stains all of the alive or dead (primary necrotic or at 100 µg/mL concentration. In addition, the cell death secondary necrotic) cells. by apoptosis was evident. The arrows in the figure show MCF-7 and MDA-MB-231 cells were seeded in a 96- pyknotic nuclei, a well-known feature of apoptosis. These well plate at the density of 1 × 104 cells per well in 200 µL results are found to be compatible with the ATP viability of culture media. After 24 h, cells were treated with plant assay. Taken together, the phase-contrast and fluorescent extracts (100 µg/mL) and then incubated for 48 h. At the images confirm the ATP assay, not the MTT assay results. end of the treatment, 2 µL of Hoechst 33342 dye was added Taking the false increments in viability into account, to each well (for 5 µg/mL final concentration). Then cells we suspected an interfering effect of the plants with the were incubated with the dye and analyzed via fluorescence MTT compound. To determine interference of the plant microscopy. extracts with the MTT compound, the plant extracts were tested in a cell-free culture system. For this purpose, the 3. Results and discussion MTT compound was added to the extract solution and Several experimental data demonstrated that the MTT then incubated for 4 h. Table 1 shows the absorbance assay yields false-positive results for viability (Ulukaya et values of plant extracts without cells. According to the al., 2004, 2008; Peng et al., 2005), and these false results led interference analysis results, the absorbance values of plant to overestimation of viability and thus underestimation of extracts were higher than the blank (with usual cell culture the cytotoxic potency of tested compounds. Therefore, in medium only) at the higher concentrations of extracts. our study we have tested the reliability of the MTT assay This clearly demonstrates the existence of interference of compared to the ATP viability assay using plant extracts plant extracts with the MTT compound. and two different cell lines. To demonstrate the differences between the MTT Figure 1 represents the viability curves of three and ATP assays, IC50 values (half-maximal inhibitory different plant extracts. It was found that viability levels concentration) were also calculated (Table 2). Based on the of the MTT assay were strikingly higher than those of MTT results, IC50 values of all plant extracts were found the ATP assay. Depending on the doses, the viability higher than 100 µg/mL in both cell lines. However, when levels were even higher than in the untreated control cells IC50 values were calculated using the ATP assay, these (100%). Surprisingly, although the cells were totally dead values were found to be lower than the MTT assay results. at especially higher concentrations, the MTT assay still These results also demonstrate that the IC50 values should produced extremely high viability values. These viability be interpreted with caution if the MTT assay is employed values were false and this finding was confirmed using when plants are concerned for cytotoxicity. both phase microscope evaluation and fluorescence Taken together, the MTT assay can suffer from imaging that confirmed cell death (Figures 2 and 3). In interference with the extracts, resulting in false-positive Figure 2, it is clearly shown that cell densities decreased results for viability. Even though in the case of total cell when both cell lines were treated with 100 µg/mL plant death, it may still give rise to increases in viability due to extracts. However, at the same concentration, the MTT the interfering (reducing) activity of the extracts with the assay did not present any cytotoxic activity. In contrast, MTT compound. it even produced an abnormally high viability value. The In conclusion, any data in the literature obtained from results of microscopic imaging were more compatible the MTT assay should be interpreted with great caution to with the ATP assay results. In fact, the ATP assay reliably avoid false-positive results for viability if plants are tested showed these cell deaths (cytotoxic effects). According to for their cytotoxic activity. these data, it should be thought that these plant extracts could interfere (reduce) with the MTT compound, leading Acknowledgment to false-positive results for viability. This study was supported by the Uludağ University Decreased cell viability (cytotoxic effect) was also Scientific Research Projects Unit (BUAP(F)-2014/3 coded shown via fluorescent microscopy (Figure 3). Therefore, project). 921 KARAKAŞ et al. / Turk J Biol MCF-7 MTT MDA-MB-231 MTT Hypericum adenotrichum ATP Hypericum adenotrichum ATP 250 250 200 200 150 150 100 100 50 50 0 0 1.56 3.13 6.25 12.5 25 50 100 1.56 3.13 6.25 12.5 25 50 100 Dose (µg/mL) Dose (µg/ml) MCF-7 MTT MDA-MB-231 MTT Salvia kronenburgii ATP Salvia kronenburgii ATP 160 160 120 120 80 80 40 40 0 0 1.56 3.13 6.25 12.5 25 50 100 1.56 3.13 6.25 12.5 25 50 100 Dose (µg/mL) Dose (µg/mL) MCF-7 MTT MDA-MB-231 MTT Pelargonium quercetorum ATP Pelargonium quercetorum ATP 160 160 120 120 80 80 40 40 0 0 1.56 3.13 6.25 12.5 25 50 100 1.56 3.13 6.25 12.5 25 50 100 Dose (µg/mL) Dose (µg/mL) Figure 1. MTT and ATP assays were used to determine the viability after treatment with three different plant extracts at different doses. Note that the MTT assay produces abnormally high viability results in comparison to the ATP assay. 922 Viability (%) Viability (%) Viability (%) Viability (%) Viability (%) Viability (%) KARAKAŞ et al. / Turk J Biol Figure 2. Phase-contrast imaging for demonstration of decrease in cell density. The cells were treated with 100 µg/mL plant extracts for 48 h, and then cells were imaged before the addition of the MTT salt. Magnification 100×. Figure 3. Fluorescence imaging to confirm cell death/apoptosis. The cells were treated with 100 µg/mL plant extracts for 48 h and then stained as explained in Section 2, followed by visualization using fluorescence microscope. Pictures show Hoechst 33342 dye staining. Arrows show the pyknotic nuclei, a well-known feature of apoptosis. Magnification 100× 923 KARAKAŞ et al. / Turk J Biol Table 1. Absorbance values of the extracts alone. H. adenotrichum, S. kronenburgii, and P. quercetorum extracts in usual culture medium were incubated with the MTT salt for 4 h in a cell-free culture system. Concentrations (µg/mL) Plant extract Blank 1.56 3.13 6.25 12.5 25 50 100 H. adenotrichum 0.053 0.017 0.034 0.068 0.135 0.271 0.542 1.084 S. kronenburgii 0.051 0.039 0.029 0.033 0.043 0.075 0.139 0.254 P. quercetorum 0.047 0.030 0.040 0.051 0.077 0.113 0.178 0.241 Table 2. IC50 values of the extracts determined by using the MTT and ATP assays. Note that great variability occurs. MCF-7 MDA-MB-231 IC50 values MTT assay ATP assay MTT assay ATP assay H. adenotrichum >100 µg/mL 10.9 µg/mL >100 µg/mL 3.1 µg/mL S. kronenburgii >100 µg/mL 22.1 µg/mL >100 µg/mL 33.0 µg/mL P. quercetorum >100 µg/mL 18.1 µg/mL >100 µg/mL 16.3 µg/mL References Alley MC, Scudiero DA, Monks A, Hursey ML, Czerwinski MJ, Fine Crouch SP, Kozlowski R, Slater KJ, Fletcher J (1993). The use of DL, Abbott BJ, Mayo JG, Shoemaker RH, Boyd MR (1988). 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