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1. Magnetic Bead-based Exosome Extraction
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	<li>Pipette a well-mixed solution of 5 &#181;L of Streptavidin-coated magnetic microparticles into 495 &#181;L of phosphate-buffered saline (PBS) buffer in a microcentrifuge tube to resuspend the beads. Wash and resuspend the beads with 500 &#181;L of PBS three times using a magnetic rack. The rack is an array of magnets on the side of a housing unit that can hold the sample microcentrifuge tubes.
	<ol>
		<li>For each wash, first, let the tubes sit on the...

magnetic bead-based exosome extraction: a technique to isolate specific exosomes from biofluids

Source: Tu, M. et al. <a href="https://www.jove.com/video/52439" target="_blank">Detection of Exosomal Biomarkers by Electric Field-induced Release and Measurement (EFIRM).</a> J. Vis. Exp. (2015)
In this video, we demonstrate a procedure to isolate exosomes from target biofluids using magnetic microbeads. The procedure enables the rapid capture of exosomes, which can be used for downstream analysis.

Magnetic Bead-based Exosome Extraction: A Technique to Isolate Specific Exosomes from Biofluids

Source: Tu, M. et al. Detection of Exosomal Biomarkers by Electric Field-induced Release and Measurement (EFIRM). J. Vis. Exp. (2015)
In this video, we ...

Exosomes are extracellular vesicular structures secreted by cells into biofluids. To capture specific exosomes from biofluids, begin by taking streptavidin-coated magnetic beads in a tube.
To this tube, add biotinylated anti-human antibodies. Biotin present on the antibodies has a high affinity for streptavidin present on the magnetic beads. This results in the conjugation of the antibodies with the magnetic beads.
Next, add a protein-rich blocking solution to the tube containing the antibody-labeled magnetic beads. The proteins in the blocking solution bind to the free sites on the magnetic beads and increase specificity of exosome binding.
Now, supplement the tube with a sample biofluid rich in exosomes. Incubate the tube with agitation. The exosomes in the biofluid display specific surface receptors, which help them bind to the antibodies attached to the surface of magnetic beads.
Next, place the tube in a magnetic field. Under the influence of magnetic field, the exosome-bound magnetic beads attach to the surface of the tube. Then, using a micropipette, aspirate the remaining biofluid from the tube.
Finally, remove the tube from the magnetic field and add a suitable buffer to elute the exosome-bound beads. These exosome-bound beads can be used for further downstream analysis.

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Research

JoVE Encyclopedia of Experiments

Cancer Research

Head and Neck Cancer

Assays & techniques

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

JoVE Journal

Biochemistry

A Modified Precipitation Method to Isolate Urinary Exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

Medicine

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

Magnetic Bead-based Exosome Extraction: A Technique to Isolate Specific Exosomes from Biofluids

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