Identifying exosome binding and internalization in blood cell subsets by imaging flow cytometry.

Only recently has the importance of extracellular vesicles as key mediators of intercellular communication been appreciated. Extracellular vesicles are membrane derived structures that include exosomes, microvesicles and apoptotic bodies. Exosomes have been shown to transfer molecules between cells and have the potential to transfer signals between cells. Exosomes are released under normal physiological conditions; however, they are also believed to serve as mediators in the pathogenesis of neurological, vascular, hematological and autoimmune diseases as well as cancer. Quantifying and characterizing exosomes in a reproducible and reliable manner has been difficult due to their small size (50 – 100 nm in diameter). Exosomes analysis can be done using high-magnification microscopy however this technique has a very low throughput. Attempts to analyze exosomes using traditional flow cytometers has been hampered by the limit of detection of such small particles and low refractive index. To overcome these limitations we have employed multispectral imaging flow cytometry that has the advantage of combining high throughput flow cytometry with higher sensitivity to small particles and the added benefit of imaging that can provide visual confirmation of particle integrity and characterization. In this study we use multispectral imaging flow cytometry to investigate the interaction of exosomes with white blood cells. Exosomes derived from different cell types will be investigated for their preferential interactions with blood cell subsets by combining immunophenotyping with morphological parameters to measure their binding and internalization.