Considerations for MESF-bead based assignment of absolute fluorescence values to nanoparticles and extracellular vesicles by flow cytometry

Flow cytometry is a promising technique to characterize nanoparticles (NPs) and extracellular vesicles (EVs). However, the majority of reported experiments, use arbitrary units to indicate fluorescence intensity. This hampers comparison of results from different laboratories and different platforms. We investigated the advised use of calibrated molecules of equivalent soluble fluorophores (MESF)-beads for assignment of absolute fluorescence to NPs and EVs. Firstly, we evaluated the use of two different FITC MESF bead sets as calibrators on three different flow cytometry platforms (BD Influx™, CytoFLEX LX™ and SORP BD FACSCelesta™). Secondly, NPs and biological 4T1 mammary carcinoma-EVs were analyzed using the BD Influx and their fluorescence signals calibrated by using different sets of FITC and PE MESF beads. Although fluorescence calibration, using bright calibrators designed for cellular flow cytometry, makes inter-platform comparison possible for fluorescently labeled cells and brightly labeled particles, but the uncertainty of the currently available calibrators, which are far out of the fluorescence range of the sub-micron particles, hampers a reliable assignment of absolute MESF numbers based on extrapolation into the dim fluorescence range. Our results illustrate the need for calibration materials specifically designed for NPs and EVs to enable a reliable assignment of absolute fluorescence values in the lower fluorescent ranges. ### Competing Interest Statement Tina Van Den Broeck and Majid Mehrpouyan are both employees of BD Biosciences, a business unit of Becton, Dickinson and Company. During the course of this study, the Wauben research group, Utrecht University, Faculty of Veterinary Medicine, Department of Biomolecular Health Sciences and BD Biosciences collaborated as a co-joined partner in the European Union Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 722148. Xiaomei Yan declares competing financial interests as a cofounder of NanoFCM Inc., a company committed to commercializing the nano-flow cytometry (nFCM) technology.