RT Journal Article
SR Electronic
T1 Intrinsic variability of fluorescence calibrators impacts the assignment of MESF or ERF values to nanoparticles and extracellular vesicles by flow cytometry
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.03.01.433358
DO 10.1101/2021.03.01.433358
A1 Lozano-Andrés, Estefanía
A1 Van Den Broeck, Tina
A1 Wang, Lili
A1 Mehrpouyan, Majid
A1 Tian, Ye
A1 Yan, Xiaomei
A1 Wauben, Marca H.M.
A1 Arkesteijn, Ger. J.A.
YR 2022
UL http://biorxiv.org/content/early/2022/11/30/2021.03.01.433358.abstract
AB Flow cytometry is commonly used to characterize nanoparticles (NPs) and extracellular vesicles (EVs) but results are often expressed in arbitrary units to indicate fluorescence intensity. This hampers interlaboratory and inter-platform comparisons. We investigated the use of molecules of equivalent soluble fluorophores (MESF)-beads for assignment of fluorescence values to NPs and EVs by comparing two FITC-MESF bead sets as calibrators on different flow cytometry platforms (BD Influx™, CytoFLEX LX™ and SORP BD FACSCelesta™). Next, fluorescence signals of NPs and EVs were calibrated using different sets of FITC and PE-MESF beads. Fluorescence calibration using beads designed for cellular flow cytometry allowed inter-platform comparison. However, the intrinsic uncertainty in the fluorescence assignment to these MESF beads impacts the reliable assignment of MESF values to NPs and EVs based on extrapolation into the dim fluorescence range. Our findings demonstrate that the use of the same set of calibration materials (vendor and lot number) and the same number of calibration points, greatly improves robust interlaboratory and inter-platform comparison of fluorescent submicron sized particles.Competing Interest StatementTina 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.