Immunophenotyping extracellular vesicles by flow cytometry using CCD-based imaging technology

Background: Extracellular vesicles are membrane derived structures that include exosomes, microvesicles and apoptotic bodies. The importance of extracellular vesicles as key mediators of intercellular communication is not well understood. Exosomes have been shown to transfer molecules between cells, potentially transmitting signals. Exosomes are released under normal physiological conditions; however, they are also believed to serve as mediators in the pathogenesis of neurological, vascular, haematological and autoimmune diseases as well as cancer. Quantifying and characterizing extracellular vesicles in a reproducible and reliable manner is challenging due to their small size (exosomes range from 30 to 100 nm in diameter). Extracellular vesicle analysis can be done using high-magnification microscopy; however, this technique has a very low throughput. Attempts to analyse extracellular vesicles using traditional PMT based flow cytometers has been hampered by the limit of detection of such small particles and their low refractive index. To overcome these limitations, we have employed the Amnis imaging technology that has the advantage of high throughput flow cytometry with higher sensitivity to small particles due to the CCD based, timedelay-integration image capturing system. Methods: Exosomes were purchased or obtained from different sources, stained with multiple labelled monoclonal antibodies and quantified by CCD-based flow cytometry. Sensitivity is calculated by standardizing each instrument to MESF standards. Results: Data will be presented using the Amnis imaging technology to immunophenotype extracellular vesicles derived from different sources. Strategies to optimize detection of extracellular vesicles will also be discussed. Summary/Conclusion: Amnis imaging technology is able to detect and phenotype exosomes with very high sensitivity.