Optimization of Magnetic Bead-Based Assay and Analysis of Macrophage-Derived Extracellular Vesicles

• Ryan Muce, Chemical Engineering, University of Delaware

• Catherine Fromen, Chemical and Biomolecular Engineering, University of Delaware
• Millicent Sullivan, Derpartment Chemical & Biomolecular Engineering, and Department of Biomedical Engineering, University of Delaware

Extracellular vesicles (EVs) are lipid membrane-bound nanoparticles that mediate intercellular communication by transporting proteins, nucleic acids, and other bioactive cargo. Their natural biocompatibility and cargo-carrying capacity make them promising candidates for therapeutic drug delivery; however, EV heterogeneity and nonspecific binding present significant challenges for reproducible detection and accurate cargo characterization. This study aimed to develop and optimize a magnetic bead-based flow cytometry assay to improve EV capture specificity and fluorescent detection. Streptavidin-coated magnetic beads were functionalized with a biotinylated anti-CD63 capture antibody, incubated with EVs, and fluorescently labeled using either Exo-FITC or PE/Cy7-conjugated anti-CD63 detection antibodies. Assay optimization focused on evaluating fluorescent labeling strategies, replacing the manufacturer’s wash buffer with Tween-20 Tris-buffered saline (T-TBS), and incorporating a 1% BSA blocking step to reduce nonspecific interactions. Flow cytometry performance was assessed using median fluorescence intensity (MFI) and separation index (SI) to quantify signal intensity and population discrimination. Preliminary results demonstrated that direct FITC labeling generated substantial nonspecific fluorescence, reducing assay specificity. Transitioning to an alternative antibody-labeling strategy improved fluorescent signal discrimination while maintaining EV detection. Replacing the proprietary wash buffer with T-TBS further reduced background fluorescence, and incorporation of a 1% BSA blocking step decreased population variability and significantly improved separation between labeled and unlabeled bead populations, producing a separation index of 2.54 despite a reduction in overall fluorescence intensity. These optimization strategies together improved assay specificity and reproducibility, resulting in cleaner flow cytometry populations and a more robust platform for EV detection and characterization. Future studies will apply the optimized assay to fluorescent cargo-tracking experiments investigating macrophage-derived EVs and nanoparticle-mediated cargo transport, enabling more reliable characterization of EV cargo dynamics and supporting the continued development of EV-based therapeutic delivery systems.