Enrichment of Extracellular Vesicle Specific Populations by Fluorescence-Activated Vesicle Sorting
Background
Given that extracellular vesicles (EVs) play an essential role as messengers determining health and disease and bear potential to serve as clinical biomarkers, it is of great interest to exclusively analyze specific EV populations. 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, from human to mice. The approach also employed different high-end fluorescent sorter cytometers to ensure feasibility.
Methods
Using density gradient ultracentrifugation or differential ultracentrifugation, we isolated two types of samples: I) EVs from blood samples of pregnant women; II) brain-derived EVs (BDEVs) from Cre-loxP transgenic mice expressing td Tomato in all the cell membranes but EGFP replacing the tdTomato fluorescence in brain endothelial cells (under the Slco1 promoter). Proper EV isolation was verified by Nanoparticle Tracking Analysis, Transmission Electron Microscopy and Western Blot. Human EVs were stained with an antibody against the Placental Alkaline Phosphatase to sort for placenta-specific EVs. The sorting was tested with BD FACSAria Fusion Sorter, BD FACSAria Illu Sorter and BD FACSDiscover S8 Cell Sorter. Subsequently, the sort was validated by Imaging Flow Cytometry (IFCM) and Liquid-chromatography coupled Tandem-Mass-Spectrometry.
Results
Trials with the different sorters revealed that both, human as well as murine cross-tissue EVs, could be successfully sorted by either Aria Fusion or Discover S8 cell sorter. Optimal parameters for nozzle size, detection thresholds, setting gates and sample collection were identified. IFCM of the sorted fractions revealed an enrichment of the populations of interest up to 80%, which was further confirmed by the detection of tissue-specific proteins in quantitative proteomic analyses.
Conclusion
We show successful sorting and enrichment of specific EVs from different sources (brain tissue, blood), species (human, mouse) using different fluorescent sorters allowing for subsequent in-depth downstream analysis.
Keywords
Sorting, FACS, EV enrichment, cross-species
Authors
Isabel Graf1 (Corresponding Author: isabel.graf[at]stud.uke.uni-hamburg[dot]de), Anne Rissiek2, Jochen Behrends3, Santra Brenna4, Christina Krüger4, Bente Siebels5, Christopher Urbschat1, Franz Ricklefs6, Anke Diemert7, Petra Arck1, Tim Magnus4, Berta Puig4 (Corresponding Author: b.puig-martorell[at]uke[dot]de)