Characterization of Extracellular Vesicle Surface Markers

The presence of CD39 and CD73 was confirmed by bead-assisted flow cytometry as previously described [18 (link)]. Briefly, 10 μg of TEX in 100 μl PBS were captured with biotin-labeled anti-CD63 mAb (1:50, Biolegend, San Diego, CA) for 2 h at RT. TEX were then incubated with 10 μL of ExoCap™ Streptavidin magnetic beads (MBL International, Woburn, MA) for 2 h at RT. The beads/anti-CD63Ab/TEX complexes were washed using a magnet and re-suspended in 200 μl of PBS. 100 μl of the sample was stained with an anti-CD39-FITC (1:25, 328206, Biolegend), anti-CD73-FITC (1:25, 344016, Biolegend) or with the appropriate isotype control for 1 h at RT. Analysis was performed with the Gallios flow cytometer and the Kaluza 1.0 software (Beckman Coulter, Krefeld, Germany). 10.000 events were acquired by gating on the bead fraction in the forward/sideward light scatter.