Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

• Type of Material: Article
• Authors: Kateena Addae Konadu, Morehouse School of Medicine; Ming Bo Huang, Morehouse School of Medicine; William Roth, Morehouse School of Medicine; Wendy Armstrong, Emory University; Michael Powell, Morehouse School of Medicine; Francois Villinger, Emory University; Vincent Bond, Morehouse School of Medicine
• Language: English
• Date: 2016-01-01
• Publisher: JoVE
• Publication Version: Final Published Version
• Copyright Statement: © 2016 Journal of Visualized Experiments.
• Final Published Version (URL): http://dx.doi.org/10.3791/53495
• Title of Journal or Parent Work: Journal of Visualized Experiments
• ISSN: 1940-087X
• Volume: 2016
• Issue: 107
• Grant/Funding Information: Kateena Addae-Konadu was supported by UNCF/Merck Graduate Research Fellowship, American Medical Association Foundation, CRECD Grant 8R25MD007589-10, and NIH NIGMS MBRS Grant R25 GM058268.; This work was supported by NIMHD grants 8G12MD007602, and 8U54MD007588, NIAID grant 1R21AI095150-01A1, Georgia Research Alliance grant GRA.VAC08.W, and Emory CFAR grant P30 A1050409.
• Keywords: Plasma; CD8(+) T-CELLS; INDUCTION; VESICLES; Velocity Gradients; Exosomes; Immunology; PROGNOSTIC VALUE; ACTIVATION MARKERS; MICROVESICLES; Science & Technology; HIV-1; Issue 107; INFECTED-CELLS; Cytokines; DISEASE PROGRESSION; NEF; APOPTOSIS; Multidisciplinary Sciences; Iodixanol
• Research Categories: Health Sciences, Pathology; Health Sciences, Immunology; Biology, Microbiology

Abstract

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated. Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge. To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.