Anti human cd63 antibody clone h5c6

Whole conditioned media, from Section 2.3, was taken directly for intact EV labelling. Conditioned media was aliquoted into 100 μl aliquots in replicates of 3 for each labelling condition (n = 3). For antibody labelling, EVs were first blocked using Human TruStain FcX (BioLegend, San Diego, CA, USA) for 10 min at RT. PE‐conjugated anti‐human antibody was added according to the optimized volumes. The anti‐human CD63 antibody (Clone H5C6; BioLegend, San Diego, CA, USA), anti‐human CD9 antibody (Clone H19a; BioLegend, San Diego, CA, USA), and anti‐human EGFR (Clone AY13; BioLegend, San Diego, CA, USA) antibodies were used for this study. IgG controls were performed using the PE‐conjugated anti‐human IgG Fc (Clone M1310G05; BioLegend, San Diego, CA, USA). CFDA‐SE labelling was performed using a 1:100 dilution of 10 mM CFDA‐SE (CFDA; Invitrogen, Waltham, MA) in dimethyl sulfoxide (DMSO; Sigma Aldrich, St. Louis, MO). Following labelling, unbound antibody and dye was eliminated via 100 kDa centrifugation filtration. The final sample was restored to 100 μl using 0.22 μm filtered 1X PBS.