Exosomes are 40-100-nm diameter membrane vesicles of endocytic origin that are released by most cell types upon fusion of multivesicular bodies with the plasma membrane, presumably as a vehicle for cell-free intercellular communication. While early studies focused on their secretion from diverse cell types in vitro, exosomes have now been identified in body fluids such as urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, breast milk, saliva and blood. Exosomes have pleiotropic biological functions, including immune response, antigen presentation, intracellular communication and the transfer of RNA and proteins. While they have also been implicated in the transport and propagation of infectious cargo, such as prions, and retroviruses, including HIV, suggesting a role in pathological situations, recent studies suggest that the presence of such infectious cargo may be artefacts of exosome-purification strategies. Improvements in mass spectrometry-based proteomic tools, both hardware and software, coupled with improved purification schemes for exosomes, has allowed more in-depth proteome analyses, contributing immensely to our understanding of the molecular composition of exosomes. Proteomic cataloguing of exosomes from diverse cell types has revealed a common set of membrane and cytosolic proteins, suggesting the evolutionary importance of these membrane particles. Additionally, exosomes express an array of proteins that reflect the originating host cell. Recent findings that exosomes contain inactive forms of both mRNA and microRNA that can be transferred to another cell and be functional in that new environment, have initiated many microRNA profiling studies of exosomes circulating in blood. These studies highlight the potential of exosomal microRNA profiles for use as diagnostic biomarkers of disease through a noninvasive blood test. The exacerbated release of exosomes in tumor cells, as evidenced by their increased levels in blood during the late stage of a disease and their overexpression of certain tumor cell biomarkers, suggests an important role of exosomes in diagnosis and biomarker studies. The aim of this article is to provide a brief overview of exosomes, including methods used to isolate and characterize exosomes. New advances in proteomic methods, and both mass spectrometry hardware and informatics tools will be covered briefly.

Exosomes: proteomic insights and diagnostic potential

Owing to the relationship between extracellular vesicles (EVs) and physiological and pathological conditions, the interest in EVs is exponentially growing. EVs hold high hopes for novel diagnostic and translational discoveries. This review provides an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs. Common misconceptions and methodological pitfalls are highlighted. Although EVs are found in all body fluids, in this review, we will focus on EVs from human blood, not only our most complex but also the most interesting body fluid for cardiovascular research.

Methodological Guidelines to Study Extracellular Vesicles.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

https://onlinelibrary.wiley.com/doi/pdf/10.1111/jth.12554

Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration.

https://www.edwinvanderpol.com/publications/manuscripts/van_der_Pol_2010_JTH_Vesicle_detection.pdf

Optical and non-optical methods for detection and characterization of microparticles and exosomes

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1538-7836.2004.00936.x

Measuring circulating cell-derived microparticles

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Measuring circulating cell-derived microparticles