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High-throughput fluorescence correlation spectroscopy analysis of extracellular vesicle markers

Extracellular vesicles (EVs) are critical delivery systems for the exchange of biological content between cells. These systems have been implicated in a myriad of biological processes including metastasis, cell-cell communication, and signaling cascades. Cell-derived vesicles (CDVs) are artificially generated from membranes of organelles within the cell and share similar properties with EVs. Proper study and characterization of both EVs and CDVs have relied heavily on the expression of unique surface proteins that can be used to identify vesicle subtypes in heterogeneous mixtures. However, current methods for identifying vesicle marker proteins are generally low throughput and lack the complexity needed to trace the cell of origin.

In a recent publication in Analytical and Bioanalytical Chemistry, Fu et al. have applied fluorescence correlation spectroscopy (FCS) toward high-throughput screening of vesicle surface components and size distribution for rapid vesicle characterization. This was done by screening heterogeneous mixtures of human embryonic kidney (HEK293) and human lung cancer (A549) derived vesicles against exosome marker antibodies to identify surface protein species. These exosome marker antibodies were labeled with CF®543 using Mix-n-Stain™ CF® Dye Antibody Labeling Kits and FCS measurements were performed on a purpose-built microscope. Relative surface protein expression was validated by western blot analysis. In addition, dynamic light scattering (DLS) analysis coupled with observed diffusion time of DiI labeled vesicles by FCS was used to measure the size distribution of isolated vesicles. Overall, the authors demonstrate that the combination of high-throughput screening with FCS allows rapid and comprehensive vesicle characterization, including size distribution, cell origin, and relative level of surface protein expression.

Learn more about our rapid and versatile Mix-N-Stain™ Antibody Labeling Kits available for labeling antibodies with CF® Dyes, HRP, R-PE, biotin, and other labels. We also offer kits for simple, no-purification labeling of small ligands and nanobodies.