Extracellular Vesicles (EVs) are potent bio-activators and inter-cellular communicators that play an important role in both health and disease. It is for this reason there is a strong interest in understanding their composition and origin, with the hope of using them as important biomarkers or therapeutics. Due to their very small size, heterogeneity, and large numbers there has been a need for better tools to measure them in an accurate and high throughput manner. While traditional flow cytometry has been widely used for this purpose, there are inherent problems with this approach, as these instruments have traditionally been developed to measure whole cells, which are orders of magnitude larger and express many more molecules of identifying epitopes.

Imaging flow cytometry, as performed with the ImagestreamX MKII, with its combination of increased fluorescence sensitivity, low background, image confirmation ability and powerful data analysis tools, provides a great tool to accurately evaluate EVs. We present here a comprehensive approach in applying this technology to the study of EVs.