fig3

Figure 3. Detection of sEV surface molecules using on-beads flow cytometry. (A) Schematic workflow of immunoprecipitation of MEG-01 sEVs using Protein A magnetic beads conjugated to anti-CD9, CD63, or CD81 antibodies; (B) SDS-PAGE with total protein staining (top panels) and western blot analysis (bottom panels) of PD and FT fractions from immunoprecipitation using anti-CD9, CD63, or CD81. EV markers CD81, HRS, and Alix are shown; (C) Schematic of control immunoprecipitation using Protein A beads pre-bound with either anti-CD63 or isotype control antibody; (D) SDS-PAGE with total protein staining and western blot of PD and FT fractions from (C), confirming specific capture of CD63-positive sEVs; (E) Workflow of bead-based flow cytometry to analyze sEV surface markers (CD9, CD63, and CD81); (F) Representative flow cytometry plots of sEV PD using CD63-coated Protein A beads, labeled with antibodies against CD9 (PE), CD63 (APC), or CD81 (PerCP-Cy5.5), along with corresponding isotype and unstained controls, and detected using flow cytometry. Representative immunoblots (B and D) and flow cytometry plots (F) from one of three independent biological experiments with similar results are shown. APC: Allophycocyanin; EV: extracellular vesicle; FT: flow-through; HRS: hepatocyte growth factor-regulated tyrosine kinase substrate; PD: pull-down; PE: phycoerythrin; PerCP-Cy5.5: peridinin-chlorophyll-protein complex-cyanine 5.5; RT: room temperature; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; sEV: small extracellular vesicle.