Chapter 3 32 Flow Cytometry (FC) is a tool to quantify and phenotype particles in suspension. However, while EVs can reach sizes up to ~8000 nm in diameter, the majority of EV are <300 nm and are therefore difficult to discriminate from background noise by conventional FC 3,24,25. In recent years, imaging flow cytometry (IFCM) has emerged as a technique that enables the discrimination and analysis of single EV. The ability of IFCM to detect submicron particles has been demonstrated by several research groups using fluorescent polystyrene beads 26-29 or the use of cell supernatant-derived EV 21. To date, several studies have reported the detection of EVs - obtained after performing isolation procedures - from plasma using IFCM 26,27,29,30. However, due to the used isolation procedures, it is difficult to evaluate whether these results represent all EVs in plasma, or if some subpopulations are missed 31. To rule out selection bias or introduction of artefacts caused by EV isolation techniques, we here demonstrate an IFCM-based methodology to phenotype and determine the concentration of human plasma-derived EVs with a diameter ≤400 nm - without prior isolation of EVs. By omitting the need for sample isolation, this method is able to directly show the status of an individual, which will be greatly beneficial in the monitoring of EVs in health and disease, and will enable researchers to explore new corners of EV biology. RESULTS Outline of the article The objective of this article is to provide an assay that will allow researchers to study single EVs directly in diluted, labeled human plasma using IFCM. The following procedures were conducted to validate our assay: size calibration of the IFCM based on scatter intensities, background analysis of the IFCM, detergent treatment of EVs, dilution experiments, and fluorescence calibration. In addition, two labeling strategies based on CFSE+Tetraspanin+ and CD9+CD31+ were evaluated by mixing human plasma with mouse plasma at different ratios. Detection of sub-micron fluorescent polystyrene beads EV analysis at the single EV level requires an instrument that is able to detect a heterogeneous sub-micron sized population. To this end, we tested the ability of
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