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

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 also tested. Both resazurin and resorufin form stable complexes with the examined β-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 β-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.