The staining method used for flow cytometry of exosomes coupled to beads was modified based on the methods described by Morales–Kastresana [36 (link)] and Theodoraki [37 (link)]. Briefly, 10 µg exosome protein was coincubated with 1 µg biotin-labeled anti-CD63 antibody (353,018, Biolegend) for 2 h at room temperature. Next, 15 µL of streptavidin-coated magnetic beads (MBL International) was added and the compounds were gently agitated on a shaker for 2 h at room temperature. The samples were washed once with dilution buffer from the kit and then coincubated with 10 µL of the detection antibody anti-PD-L1 PE (329706, Biolegend) or the labeled isotype control antibody (400314, Biolegend) for 1 h at room temperature. The complexes were resuspended in 100 µL PBS after washing them three times with dilution buffer for antigen detection (Beckman Coulter CytoFlex). The lower edge of the “positive gate” was set at the point where < 2% of the isotype control was positive.
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