Exosome Detection and Characterization:

Exosome detection and characterization based on flow cytometry.

BACKGROUND

Exosomes are small (-50-150 nm) extracellular vesicles (EVs) released from all cell types and found in body fluids and cell culture supernatants. Exosomes are generated by fusion of a specialized endosome, the multivesicular body (MVB) (1), with the plasma membrane.

Exosomes have been proposed to provide means for intercellular exchange of macromolecules, allowing the transfer of proteins, lipids, mRNA, miRNA and DNA, contributing to intercellular communication in relevant biological processes, including apoptosis, antigen presentation, angiogenesis, inflammation, and coagulation; playing therefore an important role in the development of several diseases, and specifically, modulating cancer microenvironment and the immune response (2,3).

In addition, exosomes have recently emerged as a new source of potential non-invasive biomarkers for various diseases, since they can be easily obtained from body fluids such as urine, blood, saliva or breastmilk and their composition may be directly dependent on the physiological and/or pathological state of the patient.

Even the number of secreted exosomes can change with the onset of different pathologies, so the detection of quantity variations could be of great relevance for diagnosis, especially in patients with cancer. Isolation and characterization of exosomes from body fluids can provide very valuable information for early detection, disease monitoring and development of effective treatments against cancer and autoimmune diseases, among others.

Moreover, a deeper knowledge of the exosome biology can also accelerate the use of these extracellular vesicles in fields such as regenerative medicine, vaccines and monoclonal antibodies, where they could play an important role as delivery systems, helping to increase the effectiveness of the treatments.

Research in exosomes as a potential source of biomarkers of human diseases has grown rapidly in recent years and consequently there is a large number of techniques for the isolation and characterization of this type of EVs. However, many of the current techniques are poorly standardized. Furthermore, the use of exosomes in diagnostic tests or clinical research, requires a sensitive, reproducible and high performance method for detection, characterization and quantification of exosome samples.

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    APPLICATION FIELD

    In order to overcome some of the limitations we present in the article, here we describe a specific, sensitive and easily scalable method and kit for exosome detection and characterization from supernatants of cell cultures and biological fluids by flow cytometry.

    This method is based on the use of magnetic beads coated with antibodies against tetraspanin CD63, a common marker of exosomes, which allows exosome detection in conventional cytometers. This same approach is also used by some kits already marketed by competitors, but their sensitivity of these other assays is very limited.

    Sensitivity and reproducibility of the kit is the result of the detailed study of certain critical factors, such as the volume and concentration of beads and samples, incubation times and conditions, as well as the concentration of the reagents, which was carried out during its development. Read more.