Amnis ImageStream - Analysis of individual extracellular vesicles by imaging flow cytometry

Abstract

Background & Aim Extracellular vesicles (EVs) are shed by nearly all cells and provide a novel way of intercellular communication. In addition to their increasing scientific recognition regarding their involvement in multiple biological processes, EVs of certain cell types are discussed as a novel class of biomarkers and can serve as potential therapeutic agents. Therefore, the composition of the EV containing sample reflects the heterogeneity of the origin and the EVs need to be chosen wisely dependent on the purpose of use. Due to the novelty of the field and the lack of well-established single EV analysis tools, the heterogeneity of single EVs has not been well analyzed, yet. Recently, our group revealed that imaging flow cytometry allows multi-parametric analyses of EVs at the single vesicle level. With the objective to investigate the heterogeneity of EVs in more detail, we aim to analyze EV-subpopulations from different EV containing samples (e.g. body fluids or different cell types). For the dissection, well qualified antibodies are essential because many of the tested antibodies showed a low efficiency or a background in the region of interest. Methods, Results & Conclusion Now, we have optimized the single EV analysis protocols, especially for EVs being smaller than 200 nm. By improving the gating strategy and optimizing the staining protocol for the detection of small EVs, we are currently able to dissect heterogeneity of EVs with different antibodies at a certain level. This allows us not only to determine subpopulations to question isolation or purification protocols, but also makes diagnostic statements possible.