Background/Objectives
The separation and enrichment of specific extracellular vesicle (EV) populations out of the total EV pool from tissues and peripheral blood is of fundamental interest to unravel their role in physiology, pathophysiology, and biomarker discovery. The aim of this study was to develop a highly reproducible and validated protocol for sorting cell-specific EV populations of interest across different samples, ranging from cell culture to mouse and human sources, using high-end fluorescent cell sorters.
Methods
EVs were isolated using density gradient or differential ultracentrifugation from three sample types: (I) cultured human and mouse glioblastoma cell lines, (II) brain tissue from wild-type mice, and (III) human blood samples. Isolated EVs were validated, then either stained with antibodies, labeled with fluorescent lipid dyes, or derived from genetically modified cells expressing fluorescent proteins (e.g., tdTomato). Sorting feasibility was tested on BD FACSAria Fusion, BD FACSAria III, and BD FACSDiscover S8. Sorted EVs were analyzed by Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), Imaging Flow Cytometry (IFCM), and Liquid Chromatogra- phy–Tandem Mass Spectrometry (LC-MS/MS).
Results
EVs from both murine and human sources were successfully sorted using BD FACSAria Fusion and FACSDiscover S8. Optimal nozzle size, detection thresholds, gating, and collection parameters were identified. IFCM showed enrichment of target EV populations up to 90%, including rare populations (<10% in source samples). LC-MS/MS confirmed tissue-specific protein presence in enriched frac- tions. TEM and NTA verified EV integrity and size post-sorting.
Conclusion
We demonstrated successful enrichment of specific EVs from multiple sources and species using fluorescence-activated vesicle sorting. This robust approach offers valuable potential for both basic research and translational applications.
Keywords
Extracellular vesicles, fluorescence-activated vesicle sorting, EV enrichment, Imaging Flow Cytometry, LC-MS/MS
