In Vitro Sitotoksisite Testleri

Abstract

Kimyasal ve biyolojik maddeler ya da fiziksel etkenler, hücreleri değişik derecelerde etkileyerek sitotoksisiteye yol açabilirler. Bir maddenin biyolojik davranışının anlaşılabilmesi için hücreler üzerindeki toksik ya da non-toksik etkisinin belirlenmesi gereklidir. İn vitro sitotoksisite testleri, ilaç niteliği taşıyan veya toksik profili araştırılan maddelerin değerlendirilmesi amacıyla hücre kültüründe gerçekleştirilen ölçüm metodlarıdır. Bu testlerle çok sayıda maddenin kısa sürede analiz edilmesi mümkün olmakta ve yapılacak hayvan denemeleri için temel veriler elde edilebilmektedir. Farklı mekanizmalara ve hassasiyetlere sahip çok sayıda sitotoksisite testi bulunmakla birlikte bu derlemede, araştırmacılar tarafından sıkça tercih edilen tetrazolyum testleri (MTT, MTS, XTT, WST), LDH testi, alamalar mavisi testi ve biyolüminesans testleri ele alınmıştır.

Keywords

• In vitro,Sitotoksisite,Hücre canlılık testleri

In Vitro Cytotoxicity Assays

Abstract

Chemical, biological and physical factors may cause cytotoxicity by affecting cells at different degrees. To understand the biological characteristic of a compound, it is essential to determine its toxic or non-toxic effects. In vitro cytotoxicity assays (cell viability assays) are cell culture based measuring methods that usually used for either evaluating possible drug candidates or investigating cytotoxic profiles of some compounds. It is possible to assess many compounds in a short period of time and obtain fundamental information necessary for further animal experiments through this methods. There are many cell viability assay methods exist based on colorimetric, enzymatic, luminometric principals etc that have different mechanisms and varied sensitivity. The popular assays, such as MTT, MTS, XTT, WST, LDH enzyme assay, alamar blue assay, ATP bioluminescent assay have summarized in this rewiev. 

Keywords

• In vitro,Cytotoxicity,Cell viability assays

References

1. Ahmed SA, Gogal RM Jr, Walsh JE, 1994: A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay. J Immunol Methods, 170, 211-224.
2. Altman FP, 1976: Tetrazolium salts and formazans. Prog Histochem Cytochem, 9(3), 3-6.
3. Aravinthan A, Govarthanan M, Selvam K, Praburaman L, Selvankumar T, Balamurugan R, Kamala-Kannan S, Kim JH, 2015: Sunroot mediated synthesis and characterization of silver nanoparticles and evaluation of its antibacterial and rat splenocyte cytotoxic effects. Int J Nanomedicine,1977-1983.
4. Barltrop JA, Owen TC, Cory AH, Cory JG, 1991: 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, 1, 11, 611-614.
5. Borenfreund E, Puerner JA, 1985: Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol Lett, 24(2-3), 119-124.
6. Connolly DT, Knight MB, Harakas NK, Wittwer AJ, Feder J, 1986: Determination of the number of endothelial cells in culture using an acid phosphatase assay. Anal Biochem, 152, 136-140.
7. Crouch SPM, Kozlowski R, Slater KJ, Fletcher J, 1993: The use of ATP bioluminescence as a measure cell proliferation and cytotoxicity. J Immunol Methods, 160, 81-88.
8. Decker T, Lohmann-Matthes ML, 1988: A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J Immunol Methods, 15, 61-69.

9. 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 Methods, 89, 271-277.
10. Duellman SJ, Zhou W, Meisenheimer P, Vidugiris G, Cali JJ, Gautam P, Wennerberg K, Vidugiriene J, 2015: Bioluminescent, nonlytic, real-time cell viability assay and use in inhibitor screening. Assay Drug Dev Techn,13(8), 456-465.
11. Erb RE, Ehlers MH, 1950: Resazurin reducing time as an indicator of bovine semen fertilizing capacity. J Dairy Sci, 33(12), 853-864.
12. Fan F, Wood KV, 2007: Bioluminescent assays for high-throughput screening.Assay Drug Dev Techn, 5, 1, 127-136.
13. Feoktistova M, Geserick P, Leverkus M, 2016: Crystal violet assay for determining viability of cultured cells. Cold Spring Harbor Protocol, DOI:10.1101/pdb.prot087379.
14. Galluzzi L et al, 2009: Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death Differ, 16, 1093-1107.
15. Gilbert DF, Boutros M, 2016: A protocol for a high-throughput multiplex cell viability assay. Methods Mol Biol, 1470, 75-84 Held P, 2004: Luciferase Measurements Using the Clarity™ Luminescence Microplate Reader. http://www.biotek.com/resources/articles/luciferase-measurements-plate-reader.html, Erişim tarihi; 29.12.2016.
16. Ishiyama M, Tominaga H, Shiga M, Sasamoto K, Ohkura Y, Ueno K, Watanabe M, 1995: Novel cell proliferation and cytotoxicity assays using a tetrazolium salt that produce formazan dye. In Vitro Toxicology, 8, 187-190.
17. Korzeniewski C, Callewaert DM, 1983: An enzyme-release assay for natural cyotoxicity. J Immunol Methods, 64, 313-320.
18. Lappalainen K, Jääskeläinen I, Syrjänen K, Urtti A, Syrjänen S, 1994: Comparison of cell proliferation and toxicity assays using two cationic liposomes. Pharm Res, 11(8), 1127-1131.
19. Lomakina GY, Modestoca YA, Ugarova NN, 2015: Bioluminescence assay for cell viability. Biochemistry (Mosc), 80(6), 701-713.
20. Longo-Sorbello GSA, Saydam G, Banerjee D, Bertino JR, 2006: Cytotoxicity and cell growth assays. In “Cell biology: a laboratory handbook”, Ed; Celis JE, Elseiver Academic Press, Burlington.
21. Martin A, Clynes M, 1993: Comparison of 5 microplate colorimetric assays for in vitro cytotoxicity testing and cell proliferation assays. Cytotechnology, 11, 49-58.
22. Mossman T, 1983: Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods, 65, 55-63.
23. Niles AL, Moravec RA, Hesselberth PE, Scurria MA, Daily WJ, Riss TL, 2007: A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers. Anal Biochem, 336, 2, 197-206.
24. O’Brien J, Wilson I, Orton T, Pognan F, 2000: Investigation of the alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Federation Of European Biochemical Societies, 267, 17, 5421-5426.
25. O’Connor R, Heenan M, Duffy C, Clynes M, 1998: Miniaturized in vitro methods in toxicity testing. In “Animal Cell Culture Techniques”, Ed; Clynes M, Springer Verlag, Germany.
26. Paull KD, Shoemaker RH, Boyd MR, Parsons JL, Risbood PA, Barbera WA, Sharma MN, Baker DC, Hand E, Scudiero DA, Monks A, Alley MC, Grote M, 1988: The synthesis of XTT: A new tetrazolium reagent that is bioreducible to a water-soluble formazan. J Heterocyclic Chem, 25, 911-914.
27. Petty RD, Sutherland LA, Hunter EM, 1995: Comparison of MTT and ATP-based assays for the measurement of viable cell number. Luminescence, 10, 1, 29-34.
28. Riss T, Moravec R, 2002: Introducing the CytoTox-ONETM homogeneous membrane integrity assay. Promega Notes, 82, 15-18.
29. Riss TL, Moravec RA, Niles AL, Duellmann S, Benink HA, Worzella TJ, Minor L, 2016: Assay guidance manual: cell viability assays. https://www.ncbi.nlm.nih.gov /books/NBK144065/?report=reader#!po=1.16279, Erişim tarihi; 21.12.2016.
30. Riss TL, Moravec RA, O’brien MA, Hawkins EM, Niles A, 2006: Homogeneous multiwell assays for measuring cell viability, cytotoxicity, and apoptosis. In “Handbook Of Assay Development In Drug Discovery”, Ed; Minor LK, CRC Press, Florida, USA.
31. Riss TL, Moravec RA, 2006: Cell proliferation assays: improved homogeneous methods used to measure the number of cells in culture. In “Cell Biology”, Ed; Celis JE, Elseiver Academic Press, Burlington.
32. Riss TL, Moravec RA, 2004: Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays. Assay Drug Dev Techn, 2, 1, 51-62.
33. Sali N, Nagy S, Poór M, Köszegi T, 2016: Multiparametric luminescent cell viability assay in toxicology models: A critical evaluation. J Pharmacol Toxicol Methods, 79, 45-54.
34. Scudiero DA, Shoemaker RH, Kenneth DP, Monks A, Tierney S, Nofziger TH, Currens MJ, Seniff D, Boyd MR, 1988: Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res, 48, 4827-4833.
35. Silva FSG, Starostina IG, Ivanova VV, Rizvanoz AA, Oliveira PJ, Pereira SP, 2016: Determination of metabolic viability and cell mass using a tandem resazurin/sulforhodamine B assay. Curr Protoc Toxicol, 68, 2.24.1-2.24.15 DOI: 10.1002/cptx.1.
36. Squatrito RC, Connor JP, Buller RE, 1995: Comparison of a novel redox cell growth assay to the ATP bioluminescence assay. Gynecol Oncol, 58, 101-105.
37. Tominaga H, Ishiyama M, Ohseto F, Sasamoto K, Hamamoto T, Suzuki K, Watanabe M, 1999: A water-soluble tetrazolium salt useful for colorimetric cell viability assay. Analytical Communications, 36, 47-50.
38. Verhaegen S, Coyle S,Connolly LM, O’loughlin, Clynes M, 1998: Cell cycle and cell death mechanism. In “Animal Cell Culture Techniques”, Ed; Clynes M, Springer Verlag, Germany.
39. Weyermann J, Lochmann D, Zimmer A, 2005: A practical note on the use of cytotoxicity assays. Int J Pharm, 288, 369-376.
40. Zucco F, Angelis DI, Stammati A, 1998: Cellular Models for In Vitro Toxicity Testing. In “Animal Cell Culture Techniques”, Ed; Clynes M, Springer Verlag, Germany.