IN VITRO CYTOTOXICITY TEST METHODS: MTT and NRU

IN VITRO CYTOTOXICITY TEST METHODS: MTT and NRU

The use of in vitro cytotoxicity tests is increasing day by day with the increase in human exposure to chemicals. Because these tests provide less cost and time savings than in vivo methods. The purpose of in vitro cytotoxicity tests is to detect cell viability. In this way, it is to determine the toxicity of the xenobiotic applied to the cell. In vitro cytotoxicity tests are frequently used in analyzes to detect cell viability, such as drug development or cancer research. This review focuses on MTT and NRU analysis, which are the most commonly used in vitro cytotoxicity test methods. At the same time, how in vitro cytotoxicity tests are performed, where they are used, what are the advantages and disadvantages of these tests are also reviewed in this article. The purpose of this article is to help researchers who will use the information about these frequently used tests by compiling them.

___

  • 1. Van Tonder A, Joubert AM, Cromarty AD. 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. 2015;8:1-10.
  • 2. Riss TL, Moravec RA. Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays. Assay and drug development technologies. 2004;2(1):51-62.
  • 3. Cavanaugh PF, Moskwa PS, Donish WH, Pera PJ, Richardson D, Andrese AP. A semi-automated neutral red based chemosensitivity assay for drug screening. Investigational new drugs. 1990;8:347-54.
  • 4. Eisenbrand G, Pool-Zobel B, Baker V, Balls M, Blaauboer B, Boobis A, et al. Methods of in vitro toxicology. Food and chemical toxicology. 2002;40(2-3):193-236.
  • 5. Houghton P, Fang R, Techatanawat I, Steventon G, Hylands PJ, Lee C. The sulphorhodamine (SRB) assay and other approaches to testing plant extracts and derived compounds for activities related to reputed anticancer activity. Methods. 2007;42(4):377-87.
  • 6. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, et al. New colorimetric cytotoxicity assay for anticancer-drug screening. JNCI: Journal of the National Cancer Institute. 1990;82(13):1107-12.
  • 7. Borenfreund E, Babich H, Martin-Alguacil N. Comparisons of two in vitro cytotoxicity assays—the neutral red (NR) and tetrazolium MTT tests. Toxicology in vitro. 1988;2(1):1-6.
  • 8. Martin A, Clynes M. Comparison of 5 microplate colorimetric assays for in vitro cytotoxicity testing and cell proliferation assays. Cytotechnology. 1993;11:49-58.
  • 9. Niles AL, Moravec RA, Hesselberth PE, Scurria MA, Daily WJ, Riss TL. A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers. Analytical biochemistry. 2007;366(2):197-206.
  • 10. Ferrari M, Fornasiero MC, Isetta AM. MTT colorimetric assay for testing macrophage cytotoxic activity in vitro. Journal of immunological methods. 1990;131(2):165-72.
  • 11. Oh YJ, Hong J. Application of the MTT-based colorimetric method for evaluating bacterial growth using different solvent systems. Lwt. 2022;153:112565.
  • 12. Lai C-S, Ponnusamy Y, Lim G-L, Ramanathan S. Antibacterial, antibiofilm and antibiotic-potentiating effects of a polyphenol-rich fraction of Dicranopteris linearis (Burm. f.) Underw. Journal of Herbal Medicine. 2021;25:100419.
  • 13. Grela E, Kozłowska J, Grabowiecka A. Current methodology of MTT assay in bacteria–A review. Acta histochemica. 2018;120(4):303-11.
  • 14. Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnology annual review. 2005;11:127-52.
  • 15. Ghasemi M, Turnbull T, Sebastian S, Kempson I. The MTT assay: utility, limitations, pitfalls, and interpretation in bulk and single-cell analysis. International journal of molecular sciences. 2021;22(23):12827.
  • 16. Sumantran VN. Cellular chemosensitivity assays: an overview. Cancer Cell Culture: Methods and Protocols. 2011:219-36.
  • 17. Otang WM, Grierson DS, Ndip RN. Cytotoxicity of three South African medicinal plants using the Chang liver cell line. African Journal of Traditional, Complementary and Alternative Medicines. 2014;11(2):324-9.
  • 18. Milheiro A, Nozaki K, Kleverlaan CJ, Muris J, Miura H, Feilzer AJ. In vitro cytotoxicity of metallic ions released from dental alloys. Odontology. 2016;104:136-42.
  • 19. Fotakis G, Timbrell JA. 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.
  • 20. Kumar P, Nagarajan A, Uchil PD. Analysis of cell viability by the MTT assay. Cold spring harbor protocols. 2018;2018(6):pdb. prot095505.
  • 21. Badavenkatappa gari S, Nelson VK, Peraman R. Tinospora sinensis (Lour.) Merr alkaloid rich extract induces colon cancer cell death via ROS mediated, mTOR dependent apoptosis pathway:“an in-vitro study”. BMC Complementary Medicine and Therapies. 2023;23(1):33.
  • 22. Van den Berg B. Microscopic analysis of MTT stained boar sperm cells. Open veterinary journal. 2015;5(1):58-63.
  • 23. Stockert JC, Blázquez-Castro A, Cañete M, Horobin RW, Villanueva Á. MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets. Acta histochemica. 2012;114(8):785-96.
  • 24. Wan H, Williams R, Doherty P, Williams D. A study of the reproducibility of the MTT test. Journal of Materials Science: Materials in Medicine. 1994;5:154-9.
  • 25. Petty RD, Sutherland LA, Hunter EM, Cree IA. Comparison of MTT and ATP‐based assays for the measurement of viable cell number. Journal of bioluminescence and chemiluminescence. 1995;10(1):29-34.
  • 26. Da Costa A, De Assis M, Plotkowski M. Comparative analysis of three methods to assess viability of mammalian cells in culture. Biocell: Official Journal of the Sociedades Latinoamericanas de Microscopia Electronica et al. 1999;23(1):65-72.
  • 27. Plumb JA, Milroy R, Kaye SB. Effects of the pH dependence of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer research. 1989;49(16):4435-40.
  • 28. Wang H-Z, Chang C-H, Lin C-P, Tsai M-C. Using MTT viability assay to test the cytotoxicity of antibiotics and steroid to cultured porcine corneal endothelial cells. Journal of ocular pharmacology and therapeutics. 1996;12(1):35-43.
  • 29. Bruggisser R, von Daeniken K, Jundt G, Schaffner W, Tullberg-Reinert H. Interference of plant extracts, phytoestrogens and antioxidants with the MTT tetrazolium assay. Planta medica. 2002;68(05):445-8.
  • 30. Präbst K, Engelhardt H, Ringgeler S, Hübner H. Basic colorimetric proliferation assays: MTT, WST, and resazurin. Cell viability assays: methods and protocols. 2017:1-17.
  • 31. Erkekoğlu P, BAYDAR T. Güncel in vitro sitotoksisite testleri. Hacettepe University Journal of the Faculty of Pharmacy. 2021;41(1):45-63.
  • 32. Ates G, Vanhaecke T, Rogiers V, Rodrigues RM. Assaying cellular viability using the neutral red uptake assay. Cell Viability Assays: Methods and Protocols. 2017:19-26.
  • 33. Repetto G, Del Peso A, Zurita JL. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nature protocols. 2008;3(7):1125-31.
  • 34. Borenfreund E, Puerner JA. A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90). Journal of tissue culture methods. 1985;9:7-9.
  • 35. Ceridono M, Tellner P, Bauer D, Barroso J, Alépée N, Corvi R, et al. The 3T3 neutral red uptake phototoxicity test: Practical experience and implications for phototoxicity testing–The report of an ECVAM–EFPIA workshop. Regulatory Toxicology and Pharmacology. 2012;63(3):480-8.
  • 36. De Carvalho C, Menezes P, Letenski G, Praes C, Feferman I, Lorencini M. In vitro induction of apoptosis, necrosis and genotoxicity by cosmetic preservatives: application of flow cytometry as a complementary analysis by NRU. International journal of cosmetic science. 2012;34(2):176-82.
  • 37. Cudazzo G, Smart DJ, McHugh D, Vanscheeuwijck P. Lysosomotropic-related limitations of the BALB/c 3T3 cell-based neutral red uptake assay and an alternative testing approach for assessing e-liquid cytotoxicity. Toxicology In Vitro. 2019;61:104647.
  • 38. Borenfreund E, Puerner JA. Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicology letters. 1985;24(2-3):119-24.
  • 39. Rodrigues RM, Bouhifd M, Bories G, Sacco M-G, Gribaldo L, Fabbri M, et al. Assessment of an automated in vitro basal cytotoxicity test system based on metabolically-competent cells. Toxicology in Vitro. 2013;27(2):760-7.
  • 40. Aslantürk ÖS. In vitro cytotoxicity and cell viability assays: principles, advantages, and disadvantages. Genotoxicity-A predictable risk to our actual world. 2018;2:64-80.
  • 41. Botham PA. Acute systemic toxicity. ILAR journal. 2002;43(Suppl_1):S27-S30.
  • 42. Weyermann J, Lochmann D, Zimmer A. A practical note on the use of cytotoxicity assays. International journal of pharmaceutics. 2005;288(2):369-76.
  • 43. Young FM, Phungtamdet W, Sanderson BJ. Modification of MTT assay conditions to examine the cytotoxic effects of amitraz on the human lymphoblastoid cell line, WIL2NS. Toxicology in vitro. 2005;19(8):1051-9.
  • 44. Fields W, Fowler K, Hargreaves V, Reeve L, Bombick B. Development, qualification, validation and application of the neutral red uptake assay in Chinese Hamster Ovary (CHO) cells using a VITROCELL® VC10® smoke exposure system. Toxicology in Vitro. 2017;40:144-52.