Alphascreen streptavidin coated donor beads

AlphaLISA reagents (Perkin Elmer, Inc) consisted of AlphaScreen streptavidin‐coated donor beads (6760002), AlphaLISA unconjugated‐acceptor beads (6062011), and AlphaLISA Universal buffer (AL001F). Mouse monoclonal anti‐human CD9 antibody (clone 12A12) was used to modify either the acceptor beads or biotin, following the manufacturer's protocol. AlphaLISA assay was performed in 96‐well half‐area white plates (6005560) and read on an EnSpire Alpha 2300 Multilabel Plate reader (Perkin Elmer, Inc). A 96‐well half‐area white plate was filled with 5 µL EV or 10 µL CM, 5 nmol/L biotinylated antibodies, and 50 µg/mL AlphaLISA acceptor beads conjugated to antibodies in universal buffer. The volume of each reagent was 10 µL. The plate was then incubated for 1 h at 37°C. Without going to the washing step, 25 µL of 80 µg/mL AlphaScreen streptavidin‐coated donor beads were added. The reaction mixture was incubated in the dark for another 30 min at 37°C. The, the plate was then read on an EnSpire Alpha 2300 Multilabel Plate reader at an excitation wavelength of 680 nm and emission wavelength of 615 nm. Background signals obtained from filtrated Advanced RPMI or PBS were subtracted from the measured signals.

The following antibodies were used for immunoblotting: mouse monoclonal anti-human CD63 antibody (clone H5C6, dilution 1:200) from BD Biosciences, mouse monoclonal anti-human CD9 antibody (clone ALB 6, dilution 1:200) from SantaCruz Biotechnology, mouse monoclonal anti-human CD147 antibody (clone MEM-M6/1, dilution 1:1,000) from Novus Biologicals and mouse monoclonal anti-Actin (clone C4, dilution 1:1,000) from Millipore. The secondary antibody (horseradish peroxidase-labeled sheep anti-mouse) were purchased from GE HealthCare.
The following antibodies used for ExoScreen and ELISA were developed in Shionogi & Co., LTD.: mouse monoclonal anti-human CD63 antibody (clone 8A12) and mouse monoclonal anti-human CD9 antibody (clone 12A12). Mouse monoclonal anti-human CD147 antibody (clone MEM-M6/1) was purchased from Novus Biologicals. Antibodies were used to modify either acceptor bead or biotin following the manufacturer’s protocol.
AlphaLISA reagents (Perkin Elmer, Inc., Waltham, MA 02451, USA) consisted of AlphaScreen Streptavidin-coated donor beads (6760002), AlphaLISA Unconjugated-acceptor beads (6062011) and AlphaLISA Universal buffer (AL001F). AlphaLISA assays were performed in 96-well half-area white plates (6005560) and read in an EnSpire Alpha 2300 Multilabel Plate reader (Perkin Elmer, Inc.).