Complex Formation of Resorufin and Resazurin with Β-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 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.

Cyclodextrin polymer coatings resist protein fouling, mammalian cell adhesion, and bacterial attachment

“…Alamar blue) had inherent affinity for CD, thus interfering with assay results (data not shown). Prior literature corroborates this observation 98 . Furthermore, while certain CD-based polymers can be made optically transparent 99 , the translucent PP substrates and CD polyurethane coatings in this study were not optimized for cell observation.…”

Interactions of Mycotoxin Alternariol with Cyclodextrins and Its Removal from Aqueous Solution by Beta-Cyclodextrin Bead Polymer

“…It can be explained by the fact that water molecules can significantly quench the fluorescence of aromatic fluorophores [48]. Since the inclusion of the mycotoxin by the apolar CD cavity disrupts the hydration shell of the mycotoxin, the quenching effect of water molecules decreases, and consequently the increased fluorescence signal of AOH can be observed [27,41,42]. As the modeling studies suggest, non-ionized AOH sinks deeper into the GCD than into the BCD cavity (Figure 8).…”

“…Molecular modeling studies have been performed at semi-empirical AM1 level using HyperChem 8 code [41,42]. After geometry optimization at AM1 level, the vibrational-rotational analyses were performed in harmonic approximation.…”

“…To consider the overall effect of the entropy changes, the different terms of the entropy contents of all species were calculated applying the Boltzmann-statistics. For example, after calculating the vibrational frequencies using the harmonic approximation, the entropy was determined on the common way using the following HyperChem code [41,42]:Svib=R∑i{hvi/kTe(hvikT)−1−ln[1−e(−hvikT)]} where ν i is the frequency of vibration and T is the temperature (298.16 K). The stability constants were determined from the Gibbs free energy changes associated to the complex formation at 298 K. A neutral aqueous environment was considered by the TIP3P solvation model implemented in HyperChem code (HyperChem, Hypercube Inc. 2007).…”

Development of a rapid method for assessing the efficacy of antibacterial photocatalytic coatings

“…The RGM was designed so the 'final' concentration of resazurin would be 5 µM (0.0001%w/v), similar to other published studies (e.g. Csepregi et al, 2018). This concentration ensured the use of minimal reagent while maintaining effective microbial detection.…”