Complex formation of resorufin and resazurin with b-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.