Cellular Imaging Analysis of MTT Assay Based on Tetrazolium Reduction

Yıl 2021, , 95 - 99, 28.04.2021

Mustafa Ünal Boyraz , Bestoon Shekhany , Faruk Süzergöz

https://doi.org/10.35440/hutfd.816390

Cited By: 1

Öz

Background: MTT assay is a colorimetric test to evaluate cell metabolic activity of living cells via reduction of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to insoluble formazan crystals by mitochondrial activities. The reduction of the tetrazolium dye is thought to occur by NADPH-dependent oxidoreductase enzymes in the cell cytosol. The MTT test is used to measure the cytotoxicity or cytostatic activity of generally plant and chemical compounds and toxic materials. In this study, it was aimed to monitor the uniformity of formazan formation at equal time intervals by visualizing the reduction of tetrazolium salts in cells.
Materials and Methods: In the study, K562 cells were used to observe the reduction of tetrazolium salts to MTT formazan crystals in cells. K562 cells were seeded in culture plate under sterile conditions. After adding 10 µL of 5 mg/mL MTT solution to the culture plate, the cells were incubated for 4 h at 37 °C in a humidified environment with 5% CO2. During the culture process, the cells were imaged at 15 minute-intervals for 4 hours.
Results: The behavior of viable and non-viable cells against MTT and the process of converting MTT to MTT formazan crystal by living cells were clearly monitored.
Conclusions: Visual analysis of MTT reduction directly from the incubator with image recordings at equal time intervals showed the perfect homogeneity of MTT degradation of the cells over time. With our study, we can state that the MTT test is an ideal test method for cytotoxicity research.

Anahtar Kelimeler

MTT assay, Tetrazolium salts, Formazan crystals, Reduction

Destekleyen Kurum

-

Proje Numarası

-

Teşekkür

-

Tetrazolyum İndirgemesine Dayalı MTT Testinin Hücresel Görsel Analizleri

Öz

Amaç: MTT testi, 3- (4,5-dimetiltiyazol-2-il) -2,5-difeniltetrazolyum bromürün, mitokondriyal aktivitelerle çözünmeyen formazan kristallerine indirgenmesi yoluyla canlı hücrelerin hücre metabolik aktivitesini değerlendirmek amacıyla kolorimetrik bir testtir. Tetrazolyum boya indirgenmesinin, hücre sitozolünde NADP H'ye bağımlı oksidoredüktaz enzimleri tarafından meydana getirildiği düşünülmektedir. MTT testi genellikle bitkisel ve kimyasal kökenli bileşiklerin ve toksik materyallerin sitotoksisitesini veya sitostatik aktivitesini ölçmek için kullanılmaktadır.
Materiyal ve Metod: Çalışmada, tetrazolyum tuzlarının hücrelerde MTT formazan kristallerine indirgenmesini gözlemlemek amacıyla K562 hücreleri kullanıldı. K562 hücreleri, steril şartlarda kültür plağına ekildi. Kültür plağına 10 µL 5 mg / mL MTT solüsyonu ekledikten sonra hücreler %5 CO2’ li nemli ortamda 37° C' de 4 saat inkübe edildi. Kültür işlemi sırasında hücreler, 15 dakika aralıklarla 4 saat süreyle görüntülendi.
Bulgular: Canlı ve cansız hücrelerin MTT' ye karşı davranışları ve canlı hücreler tarafından MTT' nin MTT formazan kristallerine dönüştürülmesi süreci net bir şekilde görüntülendi. Sonuç: Çalışmamızla, MTT testinin sitotoksisite araştırmaları için ideal bir test yöntemi olduğunu söyleyebiliriz.

Anahtar Kelimeler

MTT testi, Tetrazolyum tuzları, Formazan kristalleri, İndirgeme

Proje Numarası

-

Kaynakça

• 1Mossman T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods, 1983; 65:55-63.
• Korzeniewski C, Callewaert DM. An enzyme-release assay for natural cytotoxicity. J Immunol Methods, 1983; 64:313-320.
• Lü L, Zhang L, Wai MS, Yew DT, Xu J. Exocytosis of MTT formazan could exacerbate cell injury. Toxicol In Vitro. 2012; 26(4):636-44.
• Tockert JC, Blázquez-Castro A, Cañete M, Horobin RW, Villanueva A. MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets. Acta Histochem. 2012; 114:785-96.
• Tokur O, Aksoy A. In vitro sitotoksisite testleri, Harran Üniv Vet Fak Derg, 2017; 6 (1):112-118.
• Bernas T, Dobrucki JW. The role of plasma membrane in bioreduction of two tetrazolium salts, MTT, and CTC. Archives of biochemistry and biophysics, 2000; 380:108-116.
• Scudiero DA, Shoemaker RH, Paull KD, Monks A, Tierney S, Nofziger TH, 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.
• Grela E, Kozłowska J, Grabowıecka A. Current methodology of MTT assay in bacteria–A review. Acta histochemica, 2018, 120.4:303-311.
• Abzianidze V, Beltyukov P, Zakharenkova S, Moiseeva N, Mejia J, Holder A, et al. Synthesis and Biological Evaluation of Phaeosphaeride A Derivatives as Antitumor Agents. Molecules. 2018; 23:3043.
• Tian H, Yu Z. Resveratrol induces apoptosis of leukemia cell line K562 by modulation of sphingosine kinase-1 pathway. International journal of clinical and experimental pathology. 2015; 8(3):2755.
• Benov L. Effect of growth media on the MTT colorimetric assay in bacteria. PLoS One. 2019; 14(8):e0219713.
• Abud MB, Louzada RN, Isaac DLC, Souza LG, Dos Reis RG, Lima EM, et al. In vivo and in vitro toxicity evaluation of liposome-encapsulated sirolimus. Int J Retina Vitreous. 2019; 5:35.
• Nga NTH, Ngoc TTB, Trinh NTM, Thuoc TL, Thao DTP. Optimization and application of MTT assay in determining density of suspension cells. Anal Biochem. 2020; 610:113937.
• 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.
• Maleki P, Sadeghi Z, Shahryar Rahpeyma S, Taheri M, Raheb J. MTT assay dataset of polyethylenimine coated graphenoxide nanosheets on breast cancer cell lines (MCF7, MDA-MB-231, MDA-MB-468). Hum Antibodies. 2020; 28:197-202.
• Liu, Y., Peterson, D.A., Kimura, H, Schubert, D. Mechanism of Cellular 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) Reduction. Journal of Neurochemistry, 1997; 69:581-593.
• Liu, Y, Schubert, D. Cytotoxic Amyloid Peptides Inhibit Cellular 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) Reduction by Enhancing MTT Formazan Exocytosis. Journal of Neurochemistry, 1997; 69:2285-2293.
• Karakaş D, Arı 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: 919-925.
• Freımoser, Florian M. The MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities. Applied and environmental microbiology, 1999; 8: 3727-3729.
• Berridge MV, Tan AS. Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Arch Biochem Biophys. 1993; 303:474-82.