Tetrazolyum İndirgemesine Dayalı MTT Testinin Hücresel Görsel Analizleri

Abstract

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.

Keywords

• MTT testi
• Tetrazolyum tuzları
• Formazan kristalleri
• İndirgeme

Cellular Imaging Analysis of MTT Assay Based on Tetrazolium Reduction

Abstract

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.

Keywords

• MTT assay
• Tetrazolium salts
• Formazan crystals
• Reduction

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