Evaluation of Exosome Proteins by on‐Bead Flow Cytometry

摘要: Exosomes, recently re-named "small extracellular vesicles" or "sEV," are emerging as an intercellular communication system. Quantification of the molecular cargo exosomes carry by on-bead flow cytometry is needed for defining their role in information transfer and human disease. Exosomes (sEV) isolated from cell supernatants plasma cancer patients size-exclusion chromatography were captured biotinylated antibodies specific antigens exosome (e.g., tetraspanins) placed on streptavidin-labeled beads. Detection was performed with pretitered fluorochrome-labeled desired specificity. The data acquired a conventional cytometer, molecules equivalent soluble fluorochrome (MESF) beads used to quantify number fluorescent bound per bead. Isotype antibody controls obligatory. mean fluorescence intensity (MFI) value each sample converted into MESF units, separation index (SI), which quantifies stained isotype control beads, determined. Various proteins identified labeled quantified surface tumor cell-derived exosomes. To identify intravesicular cargo, such cytokines chemokines, lysed 0.3% Triton-100, lysates loaded aldehyde/sulfate latex cytometry. Examples quantitative and/or produced various cells present body fluids provided. On-bead standardized use cytometers useful method protein detection quantitation lines cancer. © 2020 International Society Advancement Cytometry.