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Anti-Cancer Agents in Medicinal Chemistry

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ISSN (Print): 1871-5206
ISSN (Online): 1875-5992

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Review Article

Different Methods for Cell Viability and Proliferation Assay: Essential Tools in Pharmaceutical Studies

Author(s): Zahra Nozhat, Mina S. Khalaji*, Mehdi Hedayati* and Sima Kheradmand Kia

Volume 22, Issue 4, 2022

Published on: 30 December, 2020

Page: [703 - 712] Pages: 10

DOI: 10.2174/1871520621999201230202614

Price: $65

Abstract

Background and Objective: The ratio of live cells to total cells in a sample is a definition for cell viability or cell toxicity. The assessment of the viable cells plays a critical role in all processes of the cell culture workflows. Overall, they are used to evaluate the survival of cells and also to optimize culture or experimental conditions following treatment with different agents or compounds, like during a drug screen. In most cases, the measurement of cell viability is the primary purpose of the experiments, for example, in pharmaceutical studies to evaluate agents' toxicity.

Methods: A literature research was conducted on cell viability assays from MEDLINE (PubMed), Web of Science and Scopus.

Results: There is a wide range of cell viability assays and different parameters such as cost, speed, and complexity of a test effect to determine the choosing method. However, each method has some advantages and disadvantages and none of them are 100% perfect.

Conclusion: Accordingly, it seems that the simultaneous utility of at least two assays will cover disadvantages to demonstrate the effects of different agents on different cell types. For instance, when one assay measures cell metabolic health, the other one checks cells permeability. Therefore, by this strategy, a researcher can report with more confidence the effective doses of the examined therapeutic agents.

Keywords: Cell viability, proliferation assay, pharmacology, MTT assay, mitochondria, metabolic.

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[1]
Tominaga, H.; Ishiyama, M.; Ohseto, F.; Sasamoto, K.; Hamamoto, T.; Suzuki, K.; Watanabe, M. A water-soluble tetrazolium salt useful for colorimetric cell viability assay. Anal. Commun., 1999, 36(2), 47-50.
[http://dx.doi.org/10.1039/a809656b]
[2]
Adan, A.; Kiraz, Y.; Baran, Y. Cell proliferation and cytotoxicity assays. Curr. Pharm. Biotechnol., 2016, 17(14), 1213-1221.
[http://dx.doi.org/10.2174/1389201017666160808160513] [PMID: 27604355]
[3]
Huyck, L.; Ampe, C.; Van Troys, M. The XTT cell proliferation assay applied to cell layers embedded in three-dimensional matrix. Assay Drug Dev. Technol., 2012, 10(4), 382-392.
[http://dx.doi.org/10.1089/adt.2011.391] [PMID: 22574651]
[4]
Stoddart, M.J. Cell viability assays: introduction. In: Mammalian cell viability; Stoddart, M.J., Ed.; Springer, 2011; 740, pp. 1-6.
[http://dx.doi.org/10.1007/978-1-61779-108-6_1]
[5]
Riss, T.; Niles, A.; Moravec, R.; Karassina, N.; Vidugiriene, J. Cytotoxicity Assays: In Vitro Methods to Measure Dead Cells. In: Assay Guidance Manual; Sittampalam, G.S.; Grossman, A.; Brimacombe, K.; Arkin, M.; Auld, D.; Austin, C.P., Eds.; Eli Lilly & Company and the National Center for Advancing Translational Sciences: Bethesda, MD, 2004.
[6]
Niles, A.L.; Moravec, R.A.; Eric Hesselberth, P.; Scurria, M.A.; Daily, W.J.; Riss, T.L. A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers. Anal. Biochem., 2007, 366(2), 197-206.
[http://dx.doi.org/10.1016/j.ab.2007.04.007] [PMID: 17512890]
[7]
Available from: https://www.promega.com/~/media/files/resources/protocols/technical%20bulletins/101/celltiter-fluor%20cell%20viability%20assay%20protocol.pdf
[8]
Borra, R.C.; Lotufo, M.A.; Gagioti, S.M.; Barros, Fde.M.; Andrade, P.M. A simple method to measure cell viability in proliferation and cytotoxicity assays. Braz. Oral Res., 2009, 23(3), 255-262.
[http://dx.doi.org/10.1590/S1806-83242009000300006] [PMID: 19893959]
[9]
McGaw, L.J.; Elgorashi, E.E.; Eloff, J.N. Cytotoxicity of African Medicinal Plants against Normal Animal and Human Cells; Elsevier, 2014, pp. 181-233.
[http://dx.doi.org/10.1016/B978-0-12-800018-2.00008-X]
[10]
Kuete, V.; Karaosmano Ylu, O.; Sivas, H. Anticancer Activities of African Medicinal Spices and Vegetables. In: Medicinal Spices and Vegetables from Africa; Kuete, V., Ed.; Academic Press, 2017; pp. 271-297.
[http://dx.doi.org/10.1016/B978-0-12-809286-6.00010-8]
[11]
Squatrito, R.C.; Connor, J.P.; Buller, R.E. Comparison of a novel redox dye cell growth assay to the ATP bioluminescence assay. Gynecol. Oncol., 1995, 58(1), 101-105.
[http://dx.doi.org/10.1006/gyno.1995.1190] [PMID: 7789873]
[12]
CellTiter-Blue® Cell Viability Assay. Available from: https://www.promega.com/~/media/files/resources/protocols/technical%20bulletins/101/celltiter-blue%20cell%20viability%20assay%20protocol.pdf
[13]
Shum, D.; Radu, C.; Kim, E.; Cajuste, M.; Shao, Y.; Seshan, V.E.; Djaballah, H. A high density assay format for the detection of novel cytotoxic agents in large chemical libraries. J. Enzyme Inhib. Med. Chem., 2008, 23(6), 931-945.
[http://dx.doi.org/10.1080/14756360701810082] [PMID: 18608772]
[14]
Pace, R.T.; Burg, K.J.L. Toxic effects of resazurin on cell cultures. Cytotechnology, 2015, 67(1), 13-17.
[http://dx.doi.org/10.1007/s10616-013-9664-1] [PMID: 24242827]
[15]
Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods, 1983, 65(1-2), 55-63.
[http://dx.doi.org/10.1016/0022-1759(83)90303-4] [PMID: 6606682]
[16]
Karakaş, D.; Ari, F.; Ulukaya, E. The MTT viability assay yields strikingly false-positive viabilities although the cells are killed by some plant extracts. Turk. J. Biol., 2017, 41(6), 919-925.
[http://dx.doi.org/10.3906/biy-1703-104] [PMID: 30814856]
[17]
Collier, A.C.; Pritsos, C.A. The mitochondrial uncoupler dicumarol disrupts the MTT assay. Biochem. Pharmacol., 2003, 66(2), 281-287.
[http://dx.doi.org/10.1016/S0006-2952(03)00240-5] [PMID: 12826270]
[18]
Ginouves, M.; Carme, B.; Couppie, P.; Prevot, G. Comparison of tetrazolium salt assays for evaluation of drug activity against Leishmania spp. J. Clin. Microbiol., 2014, 52(6), 2131-2138.
[http://dx.doi.org/10.1128/JCM.00201-14] [PMID: 24719447]
[19]
Bernas, T.; Dobrucki, J. Mitochondrial and nonmitochondrial reduction of MTT: interaction of MTT with TMRE, JC-1, and NAO mitochondrial fluorescent probes. Cytometry, 2002, 47(4), 236-242.
[http://dx.doi.org/10.1002/cyto.10080] [PMID: 11933013]
[20]
Tada, H.; Shiho, O.; Kuroshima, K.; Koyama, M.; Tsukamoto, K. An improved colorimetric assay for interleukin 2. J. Immunol. Methods, 1986, 93(2), 157-165.
[http://dx.doi.org/10.1016/0022-1759(86)90183-3] [PMID: 3490518]
[21]
Lü, L.; Zhang, L.; Wai, M.S.; Yew, D.T.; Xu, J. Exocytosis of MTT formazan could exacerbate cell injury. Toxicol. In Vitro, 2012, 26(4), 636-644.
[http://dx.doi.org/10.1016/j.tiv.2012.02.006] [PMID: 22401948]
[22]
CellTiter 96® Non-Radioactive Cell Proliferation Assay Technical Bulletin #112. Available from: http://www.promega.com/~/media/Files/Resources/Protocols/Technical%20Bulletins/0/CellTiter%2096%20Non-Radioactive%20Cell%20Proliferation%20Assay%20Protocol.pdf
[23]
Denizot, F.; Lang, R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods, 1986, 89(2), 271-277.
[http://dx.doi.org/10.1016/0022-1759(86)90368-6] [PMID: 3486233]
[24]
Hansen, M.B.; Nielsen, S.E.; Berg, K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J. Immunol. Methods, 1989, 119(2), 203-210.
[http://dx.doi.org/10.1016/0022-1759(89)90397-9] [PMID: 2470825]
[25]
Plumb, J.A.; Milroy, R.; Kaye, S.B. Effects of the pH dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer Res., 1989, 49(16), 4435-4440.
[PMID: 2743332]
[26]
Chakrabarti, R.; Kundu, S.; Kumar, S.; Chakrabarti, R. Vitamin A as an enzyme that catalyzes the reduction of MTT to formazan by vitamin C. J. Cell. Biochem., 2000, 80(1), 133-138.
[http://dx.doi.org/10.1002/1097-4644(20010101)80:1<133::AID-JCB120>3.0.CO;2-T] [PMID: 11029760]
[27]
Ishiyama, M.; Shiga, M.; Sasamoto, K.; Mizoguchi, M.; He, P-g. A new sulfonated tetrazolium salt that produces a highly water-soluble formazan dye. Chem. Pharm. Bull. (Tokyo), 1993, 41(6), 1118-1122.
[http://dx.doi.org/10.1248/cpb.41.1118]
[28]
Goodwin, C.J.; Holt, S.J.; Downes, S.; Marshall, N.J. Microculture tetrazolium assays: A comparison between two new tetrazolium salts, XTT and MTS. J. Immunol. Methods, 1995, 179(1), 95-103.
[http://dx.doi.org/10.1016/0022-1759(94)00277-4] [PMID: 7868929]
[29]
In Vitro Toxicology Assay Kit, XTT based. Available from: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/tox2bul.pdf
[30]
Scudiero, D.A.; Shoemaker, R.H.; Paull, K.D.; Monks, A.; Tierney, S.; Nofziger, T.H.; Currens, M.J.; Seniff, D.; Boyd, M.R. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res., 1988, 48(17), 4827-4833.
[PMID: 3409223]
[31]
Ameyar, M.; Wisniewska, M.; Weitzman, J.B. A role for AP-1 in apoptosis: the case for and against. Biochimie, 2003, 85(8), 747-752.
[http://dx.doi.org/10.1016/j.biochi.2003.09.006] [PMID: 14585541]
[32]
RealTime-Glo™ MT Cell Viability Assay Instructions for Use of Products G9711, G9712 and G9713 Available from: https://www.promega.com.cn/-/media/files/resources/protocols/technical-manuals/101/realtimeglo-mt-cell-viability-assay-protocol.pdf?la=en
[33]
Riss, T.L.; Moravec, R.A.; Niles, A.L.; Duellman, S.; Benink, H.A.; Worzella, T.J.; Minor, L. Cell viability assays. Assay Guidance Manual; Eli Lilly & Company and the National Center for Advancing Translational Sciences, 2016.
[34]
GarcA-a-Foncillas, J.; Sunakawa, Y.; Aderka, D.; Wainberg, Z.; Ronga, P.; Witzler, P.; Stintzing, S. Distinguishing features of cetuximab and panitumumab in colorectal cancer and other solid tumors. Front. Oncol., 2019, 9, 849.
[http://dx.doi.org/10.3389/fonc.2019.00849]
[35]
Auld, D.S.; Zhang, Y.Q.; Southall, N.T.; Rai, G.; Landsman, M.; MacLure, J.; Langevin, D.; Thomas, C.J.; Austin, C.P.; Inglese, J. A basis for reduced chemical library inhibition of firefly luciferase obtained from directed evolution. J. Med. Chem., 2009, 52(5), 1450-1458.
[http://dx.doi.org/10.1021/jm8014525] [PMID: 19215089]
[36]
Lomakina, G.Y.; Modestova, Y.A.; Ugarova, N.N. Bioluminescence assay for cell viability. Biochemistry (Mosc.), 2015, 80(6), 701-713.
[http://dx.doi.org/10.1134/S0006297915060061] [PMID: 26531016]
[37]
CellTiter-Glo® 2.0 Assay, G9241, G9242 and G9243. Available from: https://www.promega.com.cn/-/media/files/resources/protocols/technical-manuals/101/celltiterglo-2-0-assay-protocol.pdf?la=en
[38]
CellTiter-Glo® Luminescent Cell Viability AssayI G, G7571, G7572 and G7573. Available from: https://www.promega.com.cn/-/media/files/resources/protocols/technical-bulletins/0/celltiter-glo-luminescent-cell-viability-assay-protocol.pdf?la=en
[39]
Gurunathan, S.; Qasim, M.; Park, C.H.; Arsalan Iqbal, M.; Yoo, H.; Hwang, J.H.; Uhm, S.J.; Song, H.; Park, C.; Choi, Y.; Kim, J.H.; Hong, K. cytotoxicity and transcriptomic analyses of biogenic palladium nanoparticles in Human Ovarian Cancer Cells (SKOV3). Nanomaterials (Basel), 2019, 9(5), 787.
[http://dx.doi.org/10.3390/nano9050787] [PMID: 31121951]
[40]
Ashdown, C.P.; Johns, S.C.; Aminov, E.; Unanian, M.; Connacher, W.; Friend, J.; Fuster, M.M. Pulsed low-frequency magnetic fields induce tumor membrane disruption and altered cell viability. Biophys. J., 2020, 118(7), 1552-1563.
[http://dx.doi.org/10.1016/j.bpj.2020.02.013] [PMID: 32142642]
[41]
CytoTox-Glo™ Cytotoxicity Assay ,G9290, G9291 and G9292. Available from: https://www.promega.com/-/media/files/resources/protocols/technical-bulletins/101/cytotox-glo-cytotoxicity-assay-protocol.pdf?la=en
[42]
CytoTox-Fluor™, Instructions for Use Of Products G9260, G9261 AND G9262.
[43]
Ramirez, C.N.; Antczak, C.; Djaballah, H. Cell viability assessment: toward content-rich platforms. Expert Opin. Drug Discov., 2010, 5(3), 223-233.
[http://dx.doi.org/10.1517/17460441003596685] [PMID: 22823019]
[44]
Jain, A.K.; Singh, D.; Dubey, K.; Maurya, R.; Mittal, S.; Pandey, A.K. Models and Methods for In Vitro Toxicity. In: In Vitro Toxicology; Dhawan, A.; Kwon, S., Eds.; Academic Press, 2018; pp. 45-65.
[http://dx.doi.org/10.1016/B978-0-12-804667-8.00003-1]
[45]
Niles, A.L.; Moravec, R.A.; Riss, T.L. In vitro viability and cytotoxicity testing and same-well multi-parametric combinations for high throughput screening. Curr. Chem. Genomics, 2009, 3, 33-41.
[http://dx.doi.org/10.2174/1875397300903010033] [PMID: 20161834]
[46]
CytoTox-ONE™ Homogeneous Membrane Integrity Assay ,G7890, G7891 and G7892. Available from: https://www.promega.com/-/media/files/resources/protocols/technical-bulletins/101/cytotox-one-homogeneous-membrane-integrity-assay-protocol.pdf?la=en
[47]
CytoTox 96® Non-Radioactive Cytotoxicity Assay, G1780. Available from: https://www.promega.com/-/media/files/resources/protocols/technical-bulletins/0/cytotox-96-nonradioactive-cytotoxicity-assay-protocol.pdf
[48]
LDH-Glo™ Cytotoxicity Assay , J2380 AND J2381. Available from: https://www.promega.com/-/media/files/resources/protocols/technical-manuals/500/ldh-glo-cytotoxicity-assay-technical-manual.pdf?la=en
[49]
ScienCell™ LDH cytotoxicity accay , cat,8078. Available from: https://www.primarycell.com/pdf/8078.pdf
[50]
Chiaraviglio, L.; Kirby, J.E. Evaluation of impermeant, DNA-binding dye fluorescence as a real-time readout of eukaryotic cell toxicity in a high throughput screening format. Assay Drug Dev. Technol., 2014, 12(4), 219-228.
[http://dx.doi.org/10.1089/adt.2014.577] [PMID: 24831788]
[51]
Propidium iodide solid (including FluoroPure™ PI *z (P1304MP, P21493). Available from: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/mp01304.pdf
[52]
CellTox™ Green Cytotoxicity Assay, G8741, G8742, G8743 and G8731.
[53]
Majtnerová, P.; Roušar, T. An overview of apoptosis assays detecting DNA fragmentation. Mol. Biol. Rep., 2018, 45(5), 1469-1478.
[http://dx.doi.org/10.1007/s11033-018-4258-9] [PMID: 30022463]
[54]
Pfeffer, C.M.; Singh, A.T.K. Apoptosis: A target for anticancer therapy. Int. J. Mol. Sci., 2018, 19(2), E448.
[http://dx.doi.org/10.3390/ijms19020448] [PMID: 29393886]
[55]
Elmore, S. Apoptosis: A review of programmed cell death. Toxicol. Pathol., 2007, 35(4), 495-516.
[http://dx.doi.org/10.1080/01926230701320337] [PMID: 17562483]
[56]
Rottenberg, H.; Wu, S. Quantitative assay by flow cytometry of the mitochondrial membrane potential in intact cells. Biochim. Biophys. Acta, 1998, 1404(3), 393-404.
[http://dx.doi.org/10.1016/S0167-4889(98)00088-3] [PMID: 9739168]
[57]
Banfalvi, G. Methods to detect apoptotic cell death. Apoptosis, 2017, 22(2), 306-323.
[http://dx.doi.org/10.1007/s10495-016-1333-3] [PMID: 28035493]
[58]
Martinez, M.M.; Reif, R.D.; Pappas, D. Detection of apoptosis: A review of conventional and novel techniques. Anal. Methods, 2010, 2(8), 996-1004.
[http://dx.doi.org/10.1039/c0ay00247j]
[59]
van Engeland, M.; Nieland, L.J.W.; Ramaekers, F.C.S.; Schutte, B.; Reutelingsperger, C.P.M. Annexin V-affinity assay: A review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry, 1998, 31(1), 1-9.
[http://dx.doi.org/10.1002/(SICI)1097-0320(19980101)31:1<1::AID-CYTO1>3.0.CO;2-R] [PMID: 9450519]
[60]
Uphoff, A.; Hermansson, M.; Haimi, P.; Somerharju, P. Analysis of complex lipidomes. In: Medical Applications of Mass Spectrometry; VA(c)key, K.; Telekes, A.; Vertes, A., Eds.; Elsevier: Amsterdam, 2008; pp. 223-249.
[http://dx.doi.org/10.1016/B978-044451980-1.50013-6]
[61]
Boulares, A.H.; Zoltoski, A.J.; Stoica, B.A.; Cuvillier, O.; Smulson, M.E. Acetaminophen induces a caspase-dependent and Bcl-XL sensitive apoptosis in human hepatoma cells and lymphocytes. Pharmacol. Toxicol., 2002, 90(1), 38-50.
[http://dx.doi.org/10.1034/j.1600-0773.2002.900108.x] [PMID: 12005112]
[62]
Payne, A.M.; Zorman, J.; Horton, M.; Dubey, S.; ter Meulen, J.; Vora, K.A. Caspase activation as a versatile assay platform for detection of cytotoxic bacterial toxins. J. Clin. Microbiol., 2013, 51(9), 2970-2976.
[http://dx.doi.org/10.1128/JCM.01161-13] [PMID: 23824772]
[63]
Saraste, A.; Pulkki, K. Morphologic and biochemical hallmarks of apoptosis. Cardiovasc. Res., 2000, 45(3), 528-537.
[http://dx.doi.org/10.1016/S0008-6363(99)00384-3] [PMID: 10728374]
[64]
Grasl-Kraupp, B.; Ruttkay-Nedecky, B.; Koudelka, H.; Bukowska, K.; Bursch, W.; Schulte-Hermann, R. In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: A cautionary note. Hepatology, 1995, 21(5), 1465-1468.
[PMID: 7737654]
[65]
Dickey, J.S.; Redon, C.E.; Nakamura, A.J.; Baird, B.J.; Sedelnikova, O.A.; Bonner, W.M. H2AX: functional roles and potential applications. Chromosoma, 2009, 118(6), 683-692.
[http://dx.doi.org/10.1007/s00412-009-0234-4] [PMID: 19707781]
[66]
Johansson, P.; Fasth, A.; Ek, T.; Hammarsten, O. Validation of a flow cytometry-based detection of γ-H2AX, to measure DNA damage for clinical applications. Cytometry B Clin. Cytom., 2017, 92(6), 534-540.
[http://dx.doi.org/10.1002/cyto.b.21374] [PMID: 27060560]
[67]
Gerets, H.H.; Dhalluin, S.; Atienzar, F.A. Multiplexing cell viability assays; Mammalian Cell Viability; Springer, 2011, pp. 91-101.
[http://dx.doi.org/10.1007/978-1-61779-108-6_11]
[68]
ApoLive-Glo™ Multiplex Assay G6410 and G6411. Available from: https://www.promega.com.cn/-/media/files/resources/protocols/technical-manuals/101/apolive-glo-multiplex-assay-protocol.pdf?la=en
[69]
ApoTox-Glo™ Triplex Assay,G6320 and G6321. Available from: https://www.promega.com.cn/-/media/files/resources/protocols/technical-manuals/101/apotox-glo-triplex-assay-protocol.pdf?la=en

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