Rapid light-dependent degradation of fluorescent dyes in chemically-defined media

Abstract Chemically defined media is increasingly used as a tool to help standardize cell biological experiments by eliminating the variability contributed by pooled animal serum. Using chemically defined media during acquisition or collection when performing FACS can contribute to high cell viability and robust experimental design. Careful handling of fluorescently labeled samples for flow cytometric analysis is critical for reliable and repeatable results, but a thorough analysis of how chemically defined media impacts the stability of fluorochrome-antibody conjugates has not been performed. In this study, we expose fluorescent antibody-labeled cells or beads to white light in the presence of various cell culture media and provide evidence that chemically defined media permits rapid light-induced fluorescent dye degradation in a cell-independent manner. This signal loss occurs both to tandem dyes and a photostable AlexaFluor dye, and results in substantial signal degradation and fluorescence spillover into secondary detectors. Finally, we find that photostability of fluorescent dyes in chemically defined media can be restored in the presence of either serum or vitamin C, demonstrating that fluorochrome degradation in this system is mediated by the activity of reactive oxygen species. Thus, our data indicate a need to standardize chemically defined media additives to reduce the impact of oxidative stress before use in cytometric experiments.