Tumor microenvironment monitoring: isolation and flow cytometric immunophenotyping of exosomes from human plasma

Abstract:

Exosomes are small vesicles comprised of a lipid bilayer containing various proteins, RNAs and bioactive lipids. Recently, the importance of exosomes in disease has become more appreciated as research demonstrates the ability of exosomes to act as intercellular messengers, serve as biomarkers and be used as therapeutic delivery methods. Purification and enumeration of small vesicles from biological fluids often requires specialized equipment, an ultra-centrifuge and particle counter for example, which are not readily available to most clinical labs. KCAS, LLC optimized a protocol to isolate exosomes from human plasma using precipitation and characterize their phenotype using flow cytometry. We were able to characterize and enumerate exosomes from diseased patients; immunophenotype was determined by the following panel: CA125, CD59, CD9, CD63, Annexin V (phosphatidylserine), human leukocyte antigen(HLA-) A, B and C cocktail. We demonstrate that we can successfully isolate exosomes and analyze them using flow cytometry. The data provides support for clinical purification and enumeration of exosomes using common laboratory equipment as well as phenotypic analysis by flow cytometry.

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