Anti cd63

Primary antibodies anti-CD9 (clones VJ 1/10 and VJ1/20), anti-CD63 (clones TEA 3/10 and TEA 3/18 and H5C6), anti-CD81 (clone Eat2) mAbs have been previously described [13] and were purchased from Immunostep or BD Bioscience either unconjugated or as PE-conjugates. Anti-CD81 (clone 5A6) mAb was kindly provided by Dr Shoshana Levy (Stanford, USA).

Flow cytometry analysis (FACS) of EVs was performed using a commercially available ExoStep (Immunostep, Salamanca, Spain) according to the manufacturer's protocol. Briefly, EVs were captured on 6.5 μm microbeads with anti‐CD63 capture antibodies provided in the kit. The EVs‐microbead complexes were incubated with anti‐CD9 (Immunostep), anti‐CD63 (Immunostep), anti‐CD81 (Immunostep), anti‐GM130 (Novus Biologicals, Colorado, USA), and anti‐calnexin (Novus Biologicals) at 4°C for 1 h and stained by streptavidin‐phycoerythrin (PE). Mouse IgG1 antibody (Bio‐Rad, Hercules, CA, USA) was used as an isotype control. Samples were analysed by FACS (Novocyte Flow Cytometer; ACEA Bioscience, Inc., San Diego, CA, USA). Data acquisition and analysis were performed using NovoExpress software.