Perm wash buffer solution

After biotinylation with 10 mM thiolated biotin polyethylene glycol (PEG), the Au substrates were incubated in neutravidin (0.05 mg/mL, in PBS with 0.2% BSA), a linker to capture biotinylated EVs, for 1 hr. Biotinylated EVs were captured on the neutravidin-coated surface, followed by EV fixation and permeabilization in a fix/perm solution (BD Science) for 10 min. The surface passivation was achieved by placing the Au surface (with or without EVs) in a blocking solution (Superblock PBS, Thermo Fisher) for 20 min. This step is important to minimize undesired non-specific binding. The captured EVs were stained via two-step indirect labeling: firstly with a cocktail of primary antibodies (20 min) then with compatible secondary antibodies (10 min, Table S1). Thorough washing was done between steps. The EVs were labeled with fluorescently labeled streptavidin. Assay buffer was a BD perm/wash buffer solution (BD Biosciences). All antibodies used in these studies are listed in table S1. Finally, the labeled EVs were mounted with a mounting solution (Prolong Au Antifade mountant, Thermo Fisher) and covered with a glass coverslip. Fluorescence images were acquired on an Olympus BX-63 upright automated epifluorescence microscope with 40× (NA = 0.95) and 100× (NA = 1.40) objectives.

hESC-derived cells were dissociated into single cells by incubation with TrypLE for 7-10 min at 37°C and resuspended in cold FBS/PBS (5% v/v). For surface marker staining of CXCR4, 1 million cells were incubated with the directly conjugated antibody CD184/CXCR4 APC at a final dilution of 1:10 for 30 min at room temperature. For intracellular markers, 1 million cells were first fixed in 250 μl of Cytofix/Cytoprem Buffer (BD, No. 554722) for 20 min at 4°C, then washed twice with BD Perm/Wash Buffer Solution (BD, No. 554723). Cell pellet was resuspended in 80 μl FBS/BD Prem/Wash buffer (4% v/v) and incubated with the corresponding directly conjugated antibody at a final dilution of 1:80 overnight at 4°C. After incubation with the antibody, cells were washed twice and analyzed using FACSCalibur cytometer (BD Bioscience) and FlowJo software (Tree Star Inc.). Gating was defined using isotype antibodies. Details of the used antibodies are listed in supplementary table 2.