Simple liquid-chromatographic method for Nile Red quantification in cell culture in spite of photobleaching

Nile Red fluorescent marker is widely-used for different purposes, such as staining cell structures and for the visualization and localization of colloidal drug carriers. However, when fluorescence-dependent imaging or quantification is performed, the risk of inexact results is increased due to photobleaching. The proposed, simple quantification method of using an HPLC–UV–Vis system allows the determination of Nile Red even in photobleached samples. The intra- and inter-assay accuracies for all analytes were found to be within 94.9 and 100.8%, respectively, of target values. When samples underwent photobleaching by laser, UV–Vis detection varied at around 99±5%, whereas fluorescence decreased down to 86%. Such results show this method to be interesting for approaches where quantification should be performed after analysis such as fluorescent imaging.