Exosome human cd63 isolation detection kit

Culture media from hepatoma cells or primary human hepatocytes cultured with exosome-depleted serum was collected on day 4 or day 12 post-infection, respectively, and centrifuged at 1500rpm for 5 minutes. Supernatants were centrifuged at 2500rpm for 15 minutes. Media was then passed through 0.4μm and 0.2μm filters. Exosomes were precipitated by mixing with ExoQuick-TC (System Biosciences) and incubated overnight. Exosomes were pelleted and re-suspended in PBS. Exosomes were indirectly quantified by measuring total protein by BCA assay and concentrations were standardized prior to treating PBMC or CD4 T cells. For exosome treatments, PBMC or CD4 T cells were activated with anti-CD3/anti-CD28 (0.1μg/ml each) for 48 hours. Isolated exosomes (50μg) were added and cultures were incubated for 4 days. In some experiments, exosomes were further purified by incubating overnight with CD63-FITC (Biolegend;H5C6) followed by anti-FITC selection and magnetic particles (StemCell Technologies). For flow cytometry, Exosome-Human CD63 Isolation/Detection kit (Invitrogen) was used to capture exosomes, followed by staining with CD63-FITC and TGF-β-PE (IQ Products;TB21).

To detect MkExos with flow cytometry, isolated MkExos were first captured with Dynabeads magnetic beads (4.5 µm, Thermo Fisher) coated with anti-CD63 antibody at 4 °C overnight, following the manufacturing protocol in Exosome - Human CD63 Isolation/Detection kit (Invitrogen). MkMPs or bead-bound MkExos were then incubated with isotype IgG, FITC anti-CD63, or APC anti-CD81 antibodies for 15 min at room temperature before flow cytometry analysis. The latter two antibodies target the common surface antigens of endosomal origin expressed by human exosomes [26 (link)], and which are not expressed on MkMPs.