Pe mouse anti human cd63

Enriched exosomes were captured using the CD63+ Dynabead exosomes isolation kit (Invitrogen, Life Technologies #10606D). The Flow Analysis of stromal exosomes bound to Dynabeads conjugated with antibody was done according to the manufacture’s protocol. Briefly, 10 µl of exosomes (200 µg/mL) were incubated with 90 µl of CD63+ Dynabeads overnight at 4°C. Dynabead magnet was then used to positively select for bound exosomes which were then stained with PE Mouse Anti-Human CD63 (BD Bioscience, San Jose, CA). Isotype control was stained by Simultest IgG2a/IgG1 (BD Bioscience, 340394). Flow cytometry was performed on a Accuri C6 System (BD Bioscience) and analyzed on Flow Jo software.
To analyze exosomes uptaken by prostate cancer cells, exosomes were pre-labeled by PKH67 dye (Sigma), and 3000 spin columns were used to remove extra dye. The dyed exosomes were added to RPMI medium to culture cancer cells for 3 hr and then flow cytometry was performed to measure fluorescence intensity of cells.

EV analysis with flow cytometry was performed using antibody coated beads. The anti-CD63 coated beads were commercially acquired (Exosome-Human CD63 isolation/detection reagent (from cell culture media), Thermo Fisher Scientific), while the anti-STX4 and anti-SCAMP3 coated beads were made using a bead conjugation kit (Dynabeads Antibody Coupling Kit, Thermo Fisher Scientific) according to manufacturer’s instructions. Beads were washed with PBS and were then incubated with 15 μg of EVs overnight at 4 °C. After incubation, samples were washed 3 times with PBS containing 1% EV-depleted FBS followed by 15 minutes incubation with human IgG antibody at 4 °C. After another three washes, the samples were incubated for 40 minutes with primary antibodies against either CD63 (PE Mouse Anti-Human CD63, BD Pharmingen), CD81 (PE Mouse Anti-Human CD81, BD Pharmingen), STX4, or SCAMP3 diluted in PBS containing 1% EV-depleted FBS. Samples were washed and incubated for 20 minutes with Alexa 488 conjugated secondary antibodies for STX4 and SCAMP3 after which another wash was performed. Samples were run in a FACSAria (BD Pharmingen) and results were analyzed using the FlowJo software (Tri Star).