ATP-based cell viability assay is superior to trypan blue exclusion and XTT assay in measuring cytotoxicity of anticancer drugs Taxol and Imatinib,and proteasome inhibitor MG-132 on human hepatoma cell line HepG2

Abstract

BACKGROUND:

Drug-induced liver injury (DILI) is the most common reason for withdrawal of anticancer drugs from the market. To prevent adverse side effects of drugs, it is important to investigate potential toxicity in vitro. However, outcome of cytotoxicity screenings can differ remarkably depending on the method used.

OBJECTIVE:

We aimed to compare XTT, ATP-based CellTiter-Glo^®2.0 and trypan blue exclusion (TBE) assays regarding their sensitivity in detecting acute cytotoxicity on HepG2 cells after incubation with the classical anticancer drugs Taxol and Imatinib or with the proteasome inhibitor MG-132.

METHODS:

HepG2 cells were treated for 48 h and cell viability was analysed by XTT, CellTiter-Glo^®2.0 or TBE assay.

RESULTS:

All tested compounds showed a reduction of viability of HepG2 cells. However, assay results differed significantly: Both ATP-based and TBE assay showed concentration-dependent cytotoxic effects, but outcomes were less pronounced with TBE. In contrast, the widely used XTT assay did not detect any acute cytotoxicity of Taxol and Imatinib.

CONCLUSIONS:

Acute cytotoxic effects of tested compounds could be revealed. However, results were significantly different from each other with ATP assay being the most sensitive one under the conditions tested. Thus, acute cytotoxicity can be dramatically underestimated if only standard XTT test is used.

Keywords

XTT trypan blue exclusion cytotoxicity DILI HepG2 Imatinib MG-132 Taxol

1 Introduction

Many drugs are subjected to phase I and phase II biotransformation in the liver which makes the organ prone to adverse side effects by toxic drug metabolites. In fact, drug-induced liver injury (DILI) is the most common reason for drug failure during clinical phases and also the prime cause for post-market drug withdrawal [1]. The toxicity profile is therefore one of the most critical properties of a drug, and at the same time the most unpredictable characteristic, since toxic effects of a certain substance can be species-, organ- and even individual-specific [2, 3]. Induction of cell cycle arrest, proteasome inhibition or inhibition of specific oncogenic proteins such as tyrosine kinases (e.g. Bcr-Abl or Kit) are well described mechanisms of action of commonly used anticancer drugs such as Taxol [4], MG-132 [5], or Imatinib [6], respectively. Regarding the prevention of adverse side effects, it is important to investigate potential toxic effects of such agents using human in vitro liver cell systems. The gold standard for in vitro toxicity analysis is the use of freshly isolated primary human hepatocytes. This system, however, also has disadvantages such as limited tissue availability and donor variability. Thus, many hepatotoxicity studies rely on liver cell lines such as the human liver hepatoma cell line HepG2 [7 –9], HepaRG cells [10], and most recently also on Upcyte/EPCC cells, i.e. primary human hepatocytes which express certain proliferation factors [11 –13].

There are several principles to study in vitro toxicity after cell treatment, such as luminescent assays, colorimetric tetrazolium-based assays and dye exclusion assays. These commonly used methods rely either on investigation of metabolic activity of enzymes within cells, such as the XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl) -5-[(phenylamino) carbonyl] -2H-tetrazolium hydroxide) assay [14] and the ATP-based CellTiter-Glo^®2.0 assay [15, 16], whereas the trypan blue exclusion assay (TBE) can be used for analysis of cell membrane integrity. The XTT assay is a colorimetric assay, where XTT is converted to water-soluble orange coloured formazan. It is generally accepted that the XTT assay measures the pyridine nucleotide redox status of cells, i.e. the content of NADH and NADPH as parameters for metabolic activity of cells. Hence, the produced amount of formazan in a given time is proportional to the cellular metabolic activity and therefore a measure for cellular viability [17]. The CellTiter-Glo^®2.0 assay, on the other hand, is a luminescent assay: the luciferase-enzyme requires ATP to convert luciferin to oxiluciferin and energy, which is released in the form of luminescence [18]. Thus, luminescence is proportional to the amount of ATP, which serves as indicator of cellular metabolic activity and viability [19 –21]. TBE is the most common test used in in vitro studies and discriminates dead and alive cells on the basis of their membrane integrity [22].

Here, we aimed to compare the XTT assay, the ATP-based CellTiter-Glo^®2.0 assay and the TBE assay regarding their sensitivity in detecting cytotoxic effects of two classical anticancer drugs, namely Taxol and Imatinib as well as of the prototypical proteasome inhibitor MG-132 on HepG2 cells.

2 Material and methods

2.1 Cultivation of HepG2 cells

Human hepatocellular carcinoma (HepG2) cells (ATCC: HB-8065) were cultivated in Dulbecco’s MEM growth medium (Biochrom AG, Berlin, Germany) supplemented with 10% foetal bovine serum (Biochrom AG, Berlin, Germany) and 2 mM L-Alanyl-L-Glutamine (Biochrom AG, Berlin, Germany) at 37°C and 5% CO₂ in a humidified incubator.

2.2 Cell treatment

HepG2 cells were seeded in 96-well (Sarstedt, Nümbrecht, Germany) or 24-well (Sarstedt, Nümbrecht, Germany) plates as described below according to the method used. After 24 h of incubation, medium was removed from adherent cells and different concentrations (0–10μM) of Imatinib mesylate (Cayman chemical, USA), Taxol (Santa Cruz Biotechnology, Dallas, USA) or MG-132 (Calbiochem, U.S. and Canada) diluted in growth medium were added in triplicates and cells were incubated for further 48 h.

2.3 XTT and CellTiter-Glo^®2.0 assay

HepG2 cells were seeded at a density of 15000 cells/well in a total volume of 100μl in a 96-well plate. Cells were treated as described above. After 48 h, medium was carefully removed from the cells, and assay substrates mixed with fresh medium were added as follows. For the XTT assay (Roche Diagnostics, Penzberg, Germany), 75μl reaction mixture was added per well (50μl fresh growth medium supplemented with 25μl XTT labelling reagent including 1μl electron coupling reagent). The absorbance was measured after 6 h incubation at a wavelength of 490 nM using a microplate reader (FLUOstar Omega, BMG Labtech, Ortenberg, Germany). For the CellTiter-Glo^®2.0 assay (Promega, Madison, USA), 100μl reaction mixture was added per well (50μl fresh medium supplemented with 50μl of reaction substance). Further steps were performed according to the manufactures’ instructions. Shortly, cells were shaken at 350 rpm for 2 min using a rotation shaker and further incubated at room temperature for 10 min. Cells were then transferred to a white-walled 96-well plate and the luminescent signal was measured using a microplate reader.

2.4 Trypan blue exclusion (TBE) assay

Cells were seeded at a density of 130000 cells/well in a total volume of 1 ml in a 24-well plate and treated as described previously. After 48 h, supernatants were removed and cells were harvested using Trypsin/EDTA (Biochrom GmbH, Berlin, Germany) for 8 min at 37°C. Trypsin reaction was stopped with growth medium, the cell suspension was collected and centrifuged at 400 xg for 5 min. Cells were washed once with PBS (Biochrom GmbH, Berlin, Germany) and counted using a hemocytometer after addition of 10μl 0.4% Trypan blue dye (amresco, Solon, USA) to 10μl of the cell suspension.

2.5 Statistical analysis

All experimental data were expressed as relative cell viability compared to the control. Statistical differences of toxic effects on HepG2 cells were analysed using one-way ANOVA followed by Tukey’s multiple comparison test. Statistical differences regarding the methods used were analysed using two-way ANOVA followed by Dunnet’s multiple comparison test. All analyses were performed with GraphPad Prism 6.0 (GraphPad Software Inc., La Jolla, CA, USA). Results were considered as statistically significant when p < 0.05.

The paper was written in accordance with the ethical guidelines of Clinical Hemorheology and Microcirculation [23].

3 Results

We aimed to compare the sensitivity of standard cytotoxicity tests by analysing the effects of two widely used anticancer drugs and of one proteasome inhibitor on HepG2 cells. Due to the intrinsic limitations of monolayer cell cultures we focused on acute cytotoxicity analyses after short-term exposure of cells to relatively high drug dosages. Therefore, HepG2 cells were treated with either Imatinib, Taxol or MG-132 for 48 hours and cell viability was measured using the trypan blue exclusion (TBE) assay or the XTT or the ATP-based CellTiter-Glo^®2.0 assay, respectively. Results are described below for each substance tested.

3.1 Imatinib

After Imatinib treatment up to a concentration of 1μM, HepG2 cell morphology obviously was not affected compared to that of untreated cells (Fig. 1A). When 4μM of drug were applied, cell density decreased, which became even more pronounced at 10μM Imatinib treatment. Furthermore, cells rounded up at higher Imatinib concentrations (Fig. 1A).

Fig.1

Effect of Imatinib-treatment on HepG2 cells as analysed by three different methods. A) Morphology of untreated HepG2 cells and treated with 1, 4 and 10μM Imatinib for 48 h. Scale bar: 200μm; B) Relative viability of HepG2 cells after Imatinib-treatment as analysed by different methods. Asterisks directly above the bars refer to a significant reduction of cellular viability when compared to the untreated control. Asterisks above the dotted line illustrate significant differences between the methods at same Imatinib concentrations; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

The ATP-based CellTiter-Glo^®2.0 assay showed a progressive reduction of metabolic activity of HepG2 cells when incubated with Imatinib at concentrations of 4μM or 10μM for 2 days (p < 0.001; Fig. 1B). The TBE assay indicated a significant reduction (p < 0.05) in cell viability of 20% after treatment with 4μM Imatinib, which did not further decrease using a higher concentration. Interestingly, the XTT assay neither reflected the results of the ATP-based assay nor those of TBE assay, as no changes in cell viability could be seen (Fig. 1B).

A direct comparison of the methods showed that the results of the TBE and ATP-based assays after Imatinib treatment did not differ significantly up to a concentration of 4μM, but appear to be highly different (p < 0.0001) at 10μM of the drug (Fig. 1B). Results of the XTT assay differed significantly (p < 0.01) from both other assays at 4μM Imatinib. The observed difference between ATP-based assay and XTT assay further increased at a concentration of 10μM Imatinib (p < 0.0001; Fig. 1B).

3.2 Taxol

Microscopic examination in this second series of experiments with Taxol as test substance revealed that already the lowest concentration tested (0.6μM) led to cell shrinkage and rounding up of HepG2 cells, and cell density apparently decreased (Fig. 2A). This cell morphology was retained with increasing Taxol concentrations and no further changes were noticed (Fig. 2A). These observations correlated with the data given by the TBE and ATP-based assay as described below.

Fig.2

Effect of Taxol-treatment on HepG2 cells as analysed by three different methods. A) Morphology of untreated HepG2 cells and treated with 0.6, 4 and 10μM Taxol for 48 h. Scale bar: 200μm; B) Relative viability of HepG2 cells after Taxol-treatment as analysed by different methods. Asterisks directly above the bars refer to a significant reduction of cellular viability when compared to the untreated control. Asterisks above the dotted line illustrate significant differences between the methods at same Taxol concentrations; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

The TBE assay showed a significant reduction (p < 0.01) of cell viability already after treatment with 0.6μM Taxol, which did not further decrease at the higher concentrations tested (Fig. 2B). The same trend was given by the ATP-based assay; however, in this instance, cell viabilities were much lower as compared to the TBE (p < 0.0001). In contrast, the XTT assay did not correlate with these data, as no reduction of cell viability could be observed with this assay at any Taxol concentration used (Fig. 2B).

Statistical comparison of the methods confirmed that the results of the XTT assay differed remarkably from those of the ATP-based assay at all concentrations used (p < 0.0001; Fig. 2B). The trend regarding the effects of Taxol treatment as analysed with the TBE and the ATP assay was similar, but less pronounced with TBE (p < 0.001; Fig. 2B).

3.3 MG-132

MG-132 was used as third test substance on HepG2 cells. Microscopic observation clearly showed that cell numbers decreased progressively with increasing MG-132 concentrations (Fig. 3A). Morphological changes of HepG2 cells could be observed when treated with 2 or 5μM of MG-132: cells appeared rounded up and thus reduced in size, which became even more apparent at the highest concentration used (Fig. 3A).

Fig.3

Effect of MG-132-treatment on HepG2 cells as analysed by three different methods. A) Morphology of untreated HepG2 cells and treated with 0.5, 2 and 5μM MG-132 for 48 h. Scale bar: 200μm; B) Relative viability of HepG2 cells after MG-132-treatment as analysed by different methods. Asterisks directly above the bars refer to a significant reduction of cellular viability when compared to the untreated control. Asterisks above the dotted line illustrate significant differences between the methods at same MG-132 concentrations; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Treatment of cells with MG-132 showed a clear dose-dependent effect. Cell viability analysis with the ATP-based assay revealed a highly significant reduction (p < 0.0001) already after treating cells with 0.5μM MG-132 in comparison to the untreated control. When 2μM of MG-132 were applied, all three assays showed a substantial reduction (p < 0.0001) of cell viability, which further decreased at a higher concentration. However, the effect of MG-132 was most obvious with the ATP-based assay, followed by TBE (Fig. 3B).

Comparing the methods directly to each other, it became clear that results of the XTT assay were significantly different from those of the ATP-based assay when cells were treated with 0.5 or 2μM MG-132 (p < 0.0001 and p < 0.001, respectively). The outcome of the TBE assay also demonstrated a significant difference when compared to the ATP-based assay at the same concentrations (p < 0.001 and p < 0.05, respectively), but not when compared to the XTT assay (Fig. 3B).

4 Discussion

Chemotherapy is one of the famous four pillars of cancer treatment. Many chemotherapeutics are potentially cytotoxic for dividing cells; an effect which can even be increased by taking advantage of the liver first-pass effect due to the formation of phase I and phase II drug metabolites. Therefore, in vitro cytotoxicity screening using human primary cells or cell lines such as the liver hepatoma cell line HepG2 plays a crucial role when defining safety profiles of drugs. The data presented in this report show a characterisation of acute cytotoxic responses of HepG2 cells after treatment with two clinically relevant chemotherapeutics and one prototypical proteasome inhibitor using three different and commonly used cell-based assays, namely the XTT assay, The ATP-based CellTiter-Glo^®2.0 assay and the trypan blue exclusion (TBE) assay.

Tetrazolium salt (MTT, XTT, MTS, WST-1, WST-8) -based and ATP-based assays as well as TBE are widely used for preliminary screenings of cell viability in cytotoxicity studies [14 , 24–30]. However, many reports have shown that cell viability measurements for a variety of compounds do not always correlate when different cytotoxicity assays are used [31 –34]. This is in line with our results: data obtained by the three different assays gave different information on the state of cellular health.

Although data obtained from the ATP-based assay and the TBE assay were congruent regarding the trends seen (Fig. 1B–3B), the ATP-based assay always revealed stronger decreases in cell viability than the TBE assay. In contrast, the XTT assay wrongly indicated the absence of toxic effects of Taxol and Imatinib in HepG2 cells regardless of the concentration used.

It has been reported that XTT salts interfere with certain compounds during cell viability assays which in turn can result in inaccurate determination of cytotoxic effects [35 –37]. Wang et al. described that the XTT assay failed to detect decreases in cell number and viability upon treatment of CHO-K1 cells with nano-TiO₂, which is likely caused by formation of superoxide which in turn is associated with ROS formation [38]. Superoxide can reduce XTT to formazan and thus produce misleading viability results [38 –40]. Also Taxol, one of the substances used in this study, was previously associated with formation of ROS and reactive nitrogen species [41]. Therefore, it is likely that the XTT assay led to inaccurate or even absent cytotoxicity profiles in our experimental settings. Another possible reason for the insensitive outcome of the XTT assay in comparison to TBE and the ATP assay was described by Goodwin et al. [42]: they found that the XTT/PMS (N-methyl dibenzopyrazine methyl sulphate) reagent mixture was unstable and that the use of MTS, an XTT equivalent which also uses PMS as electron acceptor, should be favoured for a better reaction. In our hands, the XTT assay outcomes did not only seem to be less accurate than the other two assays used, but also did not correlate with cell morphology after Taxol and Imatinib treatment (Fig. 1A and 2A), suggesting that the XTT assay showed false negative results. This observation is supported by the work of Zwolak [43] who analysed NaVO₃ treatment on CHO-K1 cells. The XTT assay did not reproduce microscopic results in this study. Moreover, serum albumin, which is contained in commercial foetal or bovine serum preparations for routine cell culture, can lead to a concentration-dependent increase of XTT assay signals and thus to overestimation of viable cell numbers [44]. Another problem with XTT, WST-1 and also MTS is their negative charge, which should make these compounds almost unable to permeate the cell membrane; thus, formazan formation is thought to be performed at the cell surface or at the plasma membrane, but not intracellularly, due to trans-plasma membrane electron transport [24, 45].

Many toxic agents act initially at the intracellular level and changes in protein expression might require time. Therefore, membrane damage as cytotoxic effect, which is detected by the TBE assay, might not be immediately evident. It has been reported that the TBE assay trends to underestimate cellular damage, at least when compared to clonogenic assays and when analysis was performed immediately following cytotoxic treatment [46 –48]. However, toxic effects of the compounds tested in the present study were more pronounced when analysed with the TBE assay than with the XTT assay. In contrast to our findings, Zwolak [43, 49] found that TBE is less sensitive than the XTT assay to measure toxic effects of NaVO₃ on CHO-K1 cells. However, in their study, the XTT assay was outperformed by three other viability assays. Despite leading to more sensitive results than the XTT assay, the TBE assay was found to be significantly different from the ATP-based assay regarding the respective outcome. Altman et al. [50] described that the TBE assay significantly overestimated cell viability and thus underestimated cytotoxic effects. Our results also suggest that the TBE assay possibly underestimates cellular damage when compared directly to the ATP-based assay. Nonetheless, the trend of both assays was highly similar. In line with this, a good correlation between ATP and TBE assay was also reported by Kuzmits et al. [51] and Kangas et al. [52].

The ATP-based assay is a very rapid method that generates measurable signals within minutes after application [53]. In the present study, it turned out to be the most sensitive method for determination of relative cell viability after treatment with different cytostatics when compared to the TBE and XTT assay. These findings were confirmed by other authors as well, reporting that ATP-based assays are in general more sensitive than tetrazolium-based assays such as the XTT assay [54, 55].

In summary, the ATP-based, XTT and TBE assay did not lead to same or even similar results when analysing relative cell viability under constant conditions. In general, toxic effects of the compounds tested were determinable with those assays (except from Taxol and Imatinib with the XTT assay), but the results were significantly different when compared to each other. Thus, the sensitivity of the tested assays was estimated as follows: ATP > TBE > XTT. Moreover, it turned out that the XTT assay should not be used to monitor cell viability when the toxic compound of interest is associated with ROS-formation or when serum-containing cell culture medium is used. In this study on HepG2 cells, the ATP-based assay was found to be the most sensitive one for the determination of cell damage regardless the compound used. Thus, at least when testing tyrosine kinase inhibitors such as Imatinib, cytoskeleton inhibitors such as Taxol or proteasome inhibitors such as MG-132, we recommend to use ATP-based assays in first line for cytotoxicity and drug dose-response studies.

Footnotes

Acknowledgments

This work was supported by the European Fonds for Regional Development (EFRE, Brandenburg, Germany; project “Entwicklung eines physiologisch relevanten Testsystems zur In-vitro-Erfassung von Hepatotoxizität im Hochdurchsatz”; project number: 85009748) and the Gesundheitscampus Brandenburg (cluster project “Konsequenzen der Alters-assoziierten Zell- und Organfunktion”; file number: GeCa H228-05/002/008).

The work is dedicated to Prof. Friedrich Jung on occasion of his 70th birthday.

References

Onakpoya

, Heneghan

, Aronson

. Post-marketing withdrawal of 462 medicinal products because of adverse drug reactions: A systematic review of the world literature. BMC Medicine. 2016;14(1) 10. doi: 10.1186/s12916-016-0553-2

Ponsoda

, Jover

, Núñez

, Royo

, Castell

, Gómez-Lechón

. Evaluation of the cytotoxicity of 10 chemicals in human and rat hepatocytes and in cell lines: Correlation between in vitro data and human lethal concentration. Toxicology in Vitro. 1995;9(6):959–66. doi: 10.1016/0887-2333(95)00053-4

Pachkoria

, Lucena

, Molokhia

, Cueto

, Carballo

, Carvajal

, et al. Genetic and molecular factors in drug-induced liver injury: A review. Current Drug Safety. 2007;2(2):97–112. doi: 10.2174/157488607780598287

Adams

, Flora

, Goldspiel

, Wilson

, Arbuck

, Finley

. Taxol: A history of pharmaceutical development and current pharmaceutical concerns. Journal of the National Cancer Institute Monographs. 1993(15):141–7.

Cervello

, Giannitrapani

, La Rosa

, Notarbartolo

, Labbozzetta

, Poma

, et al. Induction of apoptosis by the proteasome inhibitor MG132 in human HCC cells: Possible correlation with specific caspase-dependent cleavage of beta-catenin and inhibition of beta-catenin-mediated transactivation. International Journal of Molecular Medicine. 2004;13(5):741–8. doi: 10.3892/ijmm.13.5.741

Druker

, Talpaz

, Resta

, Peng

, Buchdunger

, Ford

, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. The New England Journal of Medicine. 2001;344(14) 1031–7 doi: 10.1056/nejm200104053441401

Lahoz

, Vilá

, Fabre

, Miquel

, Rivas

, Maines

, et al. An in vitro tool to assess cytochrome P450 drug biotransformation-dependent cytotoxicity in engineered HepG2 cells generated by using adenoviral vectors. Toxicology in Vitro. 2013;27(4) 110–5 doi: 10.1016/j.tiv.2012.08.001

Schoonen

, Stevenson

, Westerink

, Horbach

. Cytotoxic effects of 109 reference compounds on rat H4IIE and human HepG2 hepatocytes. III: Mechanistic assays on oxygen consumption with MitoXpress and NAD (P) H production with Alamar Blue^TM Toxicology in Vitro. 2012;26(3):511–25. doi: 10.1016/j.tiv.2012.01.004

Seeland

, Török

, Kettiger

, Treiber

, Hafner

, Huwyler

. A cell-based, multiparametric sensor approach characterises drug-induced cytotoxicity in human liver HepG2 cells. Toxicology in Vitro. 2013;27(3) 1109–20 doi: 10.1016/j.tiv.2013.02.001

10.

Anthérieu

, Chesné

, Li

, Guguen-Guillouzo

, Guillouzo

. Optimization of the HepaRG cell model for drug metabolism and toxicity studies. Toxicology in Vitro. 2012;26(8) 1278–85 doi: 10.1016/j.tiv.2012.05.008

11.

Burkard

, Dähn

, Heinz

, Zutavern

, Sonntag-Buck

, Maltman

, et al. Generation of proliferating human hepatocytes using upcyte^® technology: Characterisation and applications in induction and cytotoxicity assays. Xenobiotica. 2012;42(10):939–56. doi: 10.3109/00498254.2012.675093

12.

Herzog

, Hansen

, Miethbauer

, Schmidtke

, Anderer

, Lupp

, et al. Primary-like human hepatocytes genetically engineered to obtain proliferation competence display hepatic differentiation characteristics in monolayer and organotypical spheroid cultures. Cell Biology International. 2016;40(3):341–53. doi: 10.1002/cbin.10574

13.

Nörenberg

, Heinz

, Scheller

, Hewitt

, Braspenning

, Ott

. Optimization of upcyte^® human hepatocytes for the in vitro micronucleus assay. Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 2013;758(1):69–79. doi: 10.1016/j.mrgentox.2013.09.008

14.

Scudiero

, Shoemaker

, Paull

, Monks

, Tierney

, Nofziger

, 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 Research. 1988;48(17):4827–33.

15.

Hitomi

, Christofferson

, Ng

, Yao

, Degterev

, Xavier

, et al. Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway. Cell. 2008;135(7) 1211–23 doi: 10.1016/j.cell.2008.10.044

16.

Arora

, Gonzales

, Hagelstrom

, Beaudry

, Choudhary

, Sima

, et al. RNAi phenotype profiling of kinases identifies potential therapeutic targets in Ewing’s sarcoma. Molecular Cancer. 2010;9:218. doi: 10.1186/1476-4598-9-218

17.

Gerlier

, Thomasset

. Use of MTT colorimetric assay to measure cell activation. Journal of Immunological Methods. 1986;94(1):57–63. doi: 10.1016/0022-1759(86)90215-2

18.

Conti

, Franks

, Brick

. Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure. 1996;4(3):287–98. doi: 10.1016/S0969-2126(96)00033-0

19.

Crouch

, Kozlowski

, Slater

, Fletcher

. The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. Journal of Immunological Methods. 1993;160(1):81–8. doi: 10.1016/0022-1759(93)90011-U

20.

Kabakov

, Gabai

. Heat-shock proteins maintain the viability of ATP-deprived cells: What is the mechanism? Trends in Cell Biology. 1994;4(6) 193–6 doi: 10.1016/0962-8924(94)90135-X

21.

DeLuca

, Wannlund

, McElroy

. Factors affecting the kinetics of light emission from crude and purified firefly luciferase. Analytical Biochemistry. 1979;95(1):194–8. doi: 10.1016/0003-2697(79)90204-5

22.

Strober

. Trypan blue exclusion test of cell viability. Current protocols in immunology. 2001; Appendix 3: Appendix 3B. doi: 10.1002/0471142735.ima03bs21

23.

Ethical guidelines for publication in Clinical Hemorheology and Microcirculation: Update 2016. Clin Hemorheol Microcirc. 2016;63(1) 1–2 doi: 10.3233/ch-162058

24.

Berridge

, Herst

, Tan

. Tetrazolium dyes as tools in cell biology: New insights into their cellular reduction. Biotechnology Annual Review. 2005;11:127–52. doi: 10.1016/s1387-2656(05)11004-7

25.

Buttke

, McCubrey

, Owen

. Use of an aqueous soluble tetrazolium/formazan assay to measure viability and proliferation of lymphokine-dependent cell lines. Journal of Immunological Methods. 1993;157(1-2):233–40. doi: 10.1016/0022-1759(93)90092-L

26.

Cory

, Owen

, Barltrop

, Cory

. Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Communications. 1991;3(7):207–12.

27.

Barltrop

, Owen

, Cory

. 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl)tetrazolium, inner salt (MTS) and related analogs of 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) reducing to purple water-soluble formazans As cell-viability indicators. Bioorganic & Medicinal Chemistry Letters. 1991;1(11):611–4. doi: 10.1016/S0960-894X(01)81162-8

28.

Galluzzi

, Aaronson

, Abrams

, Alnemri

, Andrews

, Baehrecke

, et al. Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death and Differentiation. 2009;16(8) 1093–107 doi: 10.1038/cdd.2009.44

29.

Martin

, Reutelingsperger

, McGahon

, Rader

, van Schie

, LaFace

, et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: Inhibition by overexpression of Bcl-2 and Abl. The Journal of Experimental Medicine. 1995;182(5) 1545–56 doi: 10.1084/jem.182.5.1545

30.

Kepp

, Galluzzi

, Lipinski

, Yuan

, Kroemer

. Cell death assays for drug discovery. Nature Reviews Drug Discovery. 2011;10(3):221–37. doi: 10.1038/nrd3373

31.

Weyermann

, Lochmann

, Zimmer

. A practical note on the use of cytotoxicity assays. International Journal of Pharmaceutics. 2005;288(2):369–76. doi: 10.1016/j.ijpharm.2004.09.018

32.

Fotakis

, Timbrell

. In vitro cytotoxicity assays: Comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicology Letters. 2006;160(2):171–7. doi: 10.1016/j.toxlet.2005.07.001

33.

Quent

, Loessner

, Friis

, Reichert

, Hutmacher

. Discrepancies between metabolic activity and DNA content as tool to assess cell proliferation in cancer research. Journal of Cellular and Molecular Medicine. 2010;14(4) 1003–13 doi: 10.1111/j.1582-4934.2010.01013.x

34.

Haibe-Kains

, El-Hachem

, Birkbak

, Jin

, Beck

, Aerts

, et al. Inconsistency in large pharmacogenomic studies. Nature. 2013;504(7480):389–93 doi: 10.1038/nature12831

35.

Vellonen

K-S

, Honkakoski

, Urtti

. Substrates and inhibitors of efflux proteins interfere with the MTT assay in cells and may lead to underestimation of drug toxicity. European Journal of Pharmaceutical Sciences. 2004;23(2):181–8. doi: 10.1016/j.ejps.2004.07.006

36.

Pozzolini

, Scarfi

, Benatti

, Giovine

. Interference in MTT cell viability assay in activated macrophage cell line. Anal Biochem. 2003;313(2):338–41.

37.

Bruggisser

, von Daeniken

, Jundt

, Schaffner

, Tullberg-Reinert

. Interference of plant extracts, phytoestrogens and antioxidants with the MTT tetrazolium assay. Planta Medica. 2002;68(5):445–8. doi: 10.1055/s-2002-32073

38.

Wang

, Yu

, Wickliffe

. Limitation of the MTT and XTT assays for measuring cell viability due to superoxide formation induced by nano-scale TiO2. Toxicology in vitro: An international journal published in association with BIBRA. 2011;25(8) 2147–51 doi: 10.1016/j.tiv.2011.07.007

39.

Gurr

, Wang

, Chen

, Jan

. Ultrafine titanium dioxide particles in the absence of photoactivation can induce oxidative damage to human bronchial epithelial cells. Toxicology. 2005;213(1-2):66–73. doi: 10.1016/j.tox.2005.05.007

40.

Singh

, Shi

, Duffin

, Albrecht

, van Berlo

, Hohr

, et al. Endocytosis, oxidative stress and IL-8 expression in human lung epithelial cells upon treatment with fine and ultrafine TiO Role of the specific surface area and of surface methylation of the particles. Toxicology and Applied Pharmacology. 2007;222(2):141–51. doi: 10.1016/j.taa2007.05.001

41.

Nicolson

, Conklin

. Reversing mitochondrial dysfunction, fatigue and the adverse effects of chemotherapy of metastatic disease by molecular replacement therapy. Clinical & Experimental Metastasis. 2008;25(2):161–9. doi: 10.1007/s10585-007-9129-z

42.

Goodwin

, Holt

, Downes

, Marshall

. Microculture tetrazolium assays: A comparison between two new tetrazolium salts, XTT and MTS. Journal of Immunological Methods. 1995;179(1):95–103. doi: 10.1016/0022-1759(94)00277-4

43.

Zwolak

. Comparison of five different in vitro assays for assessment of sodium metavanadate cytotoxicity in Chinese hamster ovary cells (CHO-K1 line). Toxicology and Industrial Health. 2015;31(8):677–90. doi: 10.1177/0748233713483199

44.

Instruments

B-T

, Winooski

. Serum albumin leads to false-positive results in the XTT and the MTT assay. Biotechniques. 2007;43:178–86. doi: 10.2144/000112528

45.

Lutter

A-H

, Scholka

, Richter

, Anderer

. Applying XTT, WST-1, and WST-8 to human chondrocytes: A comparison of membrane-impermeable tetrazolium salts in 2D and 3D cultures. Clinical Hemorheology and Microcirculation. 2017(Preprint):1–15 doi: 10.3233/CH-179213

46.

Roper

, Drewinko

. Comparison of in vitro methods to determine drug-induced cell lethality. Cancer Research. 1976;36(7 Part 1):2182–8.

47.

Yuhas

, Pazmino

, Proctor

, Toya

. A direct relationship between immune competence and the subcutaneous growth rate of a malignant murine lung tumor. Cancer Research. 1974;34(4):722–8.

48.

Bhuyan

, Loughman

, Fraser

, Day

. Comparison of different methods of determining cell viability after exposure to cytotoxic compounds. Experimental Cell Research. 1976;97(2):275–80. doi: 10.1016/0014-4827(76)90617-0

49.

Zwolak

. Comparison of three different cell viability assays for evaluation of vanadyl sulphate cytotoxicity in a Chinese hamster ovary K1 cell line. Toxicology and Industrial Health. 2016;32(6) 1013–25 doi: 10.1177/0748233714544190

50.

Altman

, Randers

, Rao

. Comparison of trypan blue dye exclusion and fluorometric assays for mammalian cell viability determinations. Biotechnology Progress. 1993;9(6):671–4. doi: 10.1021/bp00024a017

51.

Kuzmits

, Aiginger

, Müller

, Steurer

, Linkesch

. Assessment of the sensitivity of leukaemic cells to cytotoxic drugs by bioluminescence measurement of ATP in cultured cells. Clinical Science. 1986;71(1):81–8. doi: 10.1042/cs0710081

52.

Kangas

, Grönroos

, Nieminen

. Bioluminescence of cellular ATP: A new method for evaluating cytotoxic agents in vitro. Medical Biology. 1984;62(6):338–43.

53.

Hannah

, Beck

, Moravec

, Riss

. CellTiter-Glo^TM Luminescent cell viability assay: A sensitive and rapid method for determining cell viability. Promega Cell Notes. 2001;2:11–3.

54.

Ulukaya

, Ozdikicioglu

, Oral

, Demirci

. The MTT assay yields a relatively lower result of growth inhibition than the ATP assay depending on the chemotherapeutic drugs tested. Toxicology in Vitro. 2008;22(1):232–9. doi: 10.1016/j.tiv.2007.08.006

55.

Hamid

, Rotshteyn

, Rabadi

, Parikh

, Bullock

. Comparison of alamar blue and MTT assays for high through-put screening. Toxicology in Vitro. 2004;18(5):703–10. doi: 10.1016/j.tiv.2004.03.012

ATP-based cell viability assay is superior to trypan blue exclusion and XTT assay in measuring cytotoxicity of anticancer drugs Taxol and Imatinib,and proteasome inhibitor MG-132 on human hepatoma cell line HepG2

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1 Introduction

2 Material and methods

2.1 Cultivation of HepG2 cells

2.2 Cell treatment

2.3 XTT and CellTiter-Glo®2.0 assay

2.4 Trypan blue exclusion (TBE) assay

2.5 Statistical analysis

3 Results

3.1 Imatinib

Footnotes

Acknowledgments

References

2.3 XTT and CellTiter-Glo^®2.0 assay