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Complex Formation of Resorufin and Resazurin with ?-Cyclodextrins: Can Cyclodextrins Interfere with a Resazurin Cell Viability Assay?

ABSTRACT: Resazurin (or Alamar Blue) is a poorly fluorescent dye. During the cellular reduction of resazurin, its highly fluorescent product resorufin is formed. Resazurin assay is a commonly applied method to investigate viability of bacterial and mammalian cells. In this study, the interaction of resazurin and resorufin with ?-cyclodextrins was investigated employing spectroscopic and molecular modeling studies. Furthermore, the influence of ?-cyclodextrins on resazurin-based cell viability assay was also tested. Both resazurin and resorufin form stable complexes with the examined ?-cyclodextrins (2.0-3.1 × 10³ and 1.3-1.8 × 10³ L/mol were determined as binding constants, respectively). Cells were incubated for 30 and 120 min and treated with resazurin and/or ?-cyclodextrins. Our results suggest that cyclodextrins are able to interfere with the resazurin-based cell viability assay that presumably results from the following mechanisms: (1) inhibition of the cellular uptake of resazurin and (2) enhancement of the fluorescence signal of the formed resorufin.

SUBMITTER: Csepregi R

PROVIDER: S-EPMC6017802 | biostudies-other | 2018 Feb

REPOSITORIES: biostudies-other

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Complex Formation of Resorufin and Resazurin with Β-Cyclodextrins: Can Cyclodextrins Interfere with a Resazurin Cell Viability Assay?

Csepregi Rita R Lemli Beáta B Kunsági-Máté Sándor S Szente Lajos L Kőszegi Tamás T Németi Balázs B Poór Miklós M

Molecules (Basel, Switzerland) 20180210 2

Resazurin (or Alamar Blue) is a poorly fluorescent dye. During the cellular reduction of resazurin, its highly fluorescent product resorufin is formed. Resazurin assay is a commonly applied method to investigate viability of bacterial and mammalian cells. In this study, the interaction of resazurin and resorufin with β-cyclodextrins was investigated employing spectroscopic and molecular modeling studies. Furthermore, the influence of β-cyclodextrins on resazurin-based cell viability assay was al ...[more]

PMID: 29439432

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Project description:This study evaluated in vitro cell viability by the colorimetric MTT stands for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay compared to image analysis by CellProfiler® software.Hepatoma (Hepa-1c1c7) and fibroblast (L929) cells were exposed to isolated substances, camptothecin, lycorine, tazettine, albomaculine, 3-epimacronine, trispheridine, galanthine and Padina gymnospora, Sargassum sp. methanolic extract, and Habranthus itaobinus Ravenna ethyl acetate in different concentrations. After MTT assay, cells were stained with Panotic dye kit. Cell images were obtained with an inverted microscope equipped with a digital camera. The images were analyzed by CellProfiler®.No cytotoxicity at the highest concentration analyzed for 3-epimacronine, albomaculine, galanthine, trispheridine, P. gymnospora extract and Sargassum sp. extract where detected. Tazettine offered cytotoxicity only against the Hepa1c1c7 cell line. Lycorine, camptothecin, and H. itaobinus extract exhibited cytotoxic effects in both cell lines. The viability methods tested were correlated demonstrated by Bland-Atman test with normal distribution with mean difference between the two methods close to zero, bias value 3.0263. The error was within the limits of the confidence intervals and these values had a narrow difference. The correlation between the two methods was demonstrated by the linear regression plotted as R2.CellProfiler® image analysis presented similar results to the MTT assay in the identification of viable cells, and image analysis may assist part of biological analysis procedures. The presented methodology is inexpensive and reproducible.In vitro cell viability assessment with MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay may be replaced by image analysis by CellProfiler®. The viability methods tested were correlated demonstrated by Bland-Atman test with normal distribution with mean difference between the two methods close to zero, bias value 3.0263. The correlation between the two methods was demonstrated by the linear regression plotted as R2. Abbreviations: HPLC: High pressure liquid chromatography MTT: (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide).

Dataset Information

Complex Formation of Resorufin and Resazurin with ?-Cyclodextrins: Can Cyclodextrins Interfere with a Resazurin Cell Viability Assay?

Publications

Complex Formation of Resorufin and Resazurin with Β-Cyclodextrins: Can Cyclodextrins Interfere with a Resazurin Cell Viability Assay?

Similar Datasets

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