Sector instrument

EV surface markers were assayed using prototype S-PLEX ultrasensitive assays (Meso Scale Discovery). Each S-PLEX plates was coated with CD9, CD81, CD63 capture antibodies and one isotype IgG1 antibody. bdEV samples were diluted 20-fold and incubated with the plates at RT with continuous shaking. bdEVs captured by each antibody spot were detected by a cocktail of antibodies targeting CD9, CD81, and CD63. Assay plates were then read with MSD GOLD Read buffer B on an MSD SECTOR instrument. Signal from IgG1 isotype control and Dulbecco's phosphate-buffered saline were subsequently subtracted from signals on each detection antibody capture spot before further analysis.

EV surface markers were assayed using prototype S-PLEX^® ultrasensitive assays on intact EVs. Each U-PLEX^® 96-well plates were coated with nine capture antibodies and one isotype IgG1 control antibody. Four different multiplexed assay panels (as listed in Supplementary Table 4) were used in this study. EV samples were diluted 20-fold and added to the plates incubated at RT with continuous shaking. EVs captured by each antibody spot were detected using MSD’s S-PLEX^® ultrasensitive assay methods with a cocktail of detection antibodies targeting CD63, CD81, and CD9. Assay plates were then read with MSD GOLD™ Read buffer B on an MSD^® SECTOR instrument. The ECL signal from DPBS and IgG1 isotype control were subtracted from signals on each detection antibody capture spot before further analysis.