期刊
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
卷 -, 期 107, 页码 -出版社
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/53495
关键词
Immunology; Issue 107; Exosomes; HIV-1; Plasma; Iodixanol; Velocity Gradients; Cytokines
资金
- UNCF/Merck Graduate Research Fellowship
- American Medical Association Foundation
- CRECD [8R25MD007589-10]
- NIH NIGMS MBRS [R25 GM058268]
- NIMHD [8G12MD007602, 8U54MD007588]
- NIAID [1R21AI095150-01A1]
- Georgia Research Alliance [GRA.VAC08.W]
- Emory CFAR [P30 A1050409]
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [R21AI095150] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R25GM058268] Funding Source: NIH RePORTER
- National Institute on Minority Health and Health Disparities [G12MD007602, U54MD007588, R25MD007589] Funding Source: NIH RePORTER
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.
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