Name: isolation and analysis of traceable and functionalized extracellular vesicles from the plasma and solid tissues
Uploaded: 2022-10-17T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues at JoVE.com

This work was supported by grants from the National Natural Science Foundation of China (32000974, 81870796, 82170988, and 81930025) and the China Postdoctoral Science Foundation (2019M663986 and BX20190380). We are grateful for the assistance of the National Experimental Teaching Demonstration Center for Basic Medicine (AMFU).

The animal experiments were performed in accordance with the Guidelines of Institutional Animal Care and Use Committee of the Fourth Military Medical University and the ARRIVE guidelines. For the present study, 8-week-old C57Bl/6 mice (no preference for either females or males) were used. The steps involved in isolating plasma and tissue EVs are illustrated in Figure 1. The plasma is taken as a representative to describe the EV isolation procedure from body fluids. The maxillary bone is take...

<ol>
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When studying the features, the fate, and the function of EVs, it is crucial to isolate EVs with high yield and low contamination. Various methods exist to extract EVs, such as density gradient centrifugation (DGC), size-exclusion chromatography (SEC), and immunocapture assays4,20. Here, one of the most commonly used methods, differential centrifugation, was used; the advantages of this are that it is not time consuming, it generates a high yield of EVs with easy...

Extracellular vesicles (EVs) have been proposed to define cell-released lipid bilayer-enclosed extracellular structures1, which play important roles in various physiological and pathological events2. EVs released by healthy cells can be broadly divided into two main categories, namely exosomes (or small EVs) formed through an intracellular endocytic trafficking pathway3 and microvesicles (or large EVs) developed by the outward budding of the plasma membrane of the cell4. While many studies focus on the function of EVs collected from cultured cells in vitro5, EVs derived from the circulation or tissues are more complex and heterogeneous, which have the advantage of reflecting the true state of the organism in vivo6. Furthermore, nearly all kinds of tissues can produce EVs in vivo, and these EVs can act as messengers within the tissue or be transferred by various body fluids, especially the peripheral blood, to facilitate systemic communication7. EVs in the circulation and tissues are also targets for disease diagnosis and treatment8.
Whereas exosomes have been intensively studied in recent years, microvesicles also have important biological functions, which can be easily extracted without ultracentrifugation, thus promoting basic and clinical research9. Notably, a critical issue regarding EVs isolated from the circulation and tissues is that they are derived from different cell types10. Since microvesicles are blebbed from the plasma membrane and featured by an abundance of cell surface molecules9, using parent cell membrane markers to identify the cellular origin of these EVs is feasible. Specifically, the flow cytometry (FC) technique can be applied to detect membrane markers. Moreover, researchers can isolate the EVs and make further analyses based on the functional cargos.
The present protocol provides a thorough procedure for extracting and characterizing EVs from in vivo samples. The EVs are isolated via differential centrifugation, and the characterization of EVs includes morphological identification via nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), origin analysis via FC, and protein cargo analysis via western blot. The blood plasma and maxillary bone of mice are used as representatives. Researchers can refer to this protocol for EVs from other sources and make corresponding modifications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4% paraformaldehyde&#160;</td>
			<td>Biosharp</td>
			<td>143174</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>Alexa fluor 488 anti-goat secondary antibody</td>
			<td>Yeason</td>
			<td>34306ES60</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Alexa fluor 488 anti-rabbit secondary antibody</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Anti-CD18 antibody</td>
			<td>Abcam</td>
			<td>ab131044</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Anti-CD81 antibody</td>
			<td>Abcam</td>
			<td>ab109201</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>anti-CD9 antibody</td>
			<td>Huabio</td>
			<td>ET1601-9</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Anti-Mitofilin antibody</td>
			<td>Abcam</td>
			<td>ab110329</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>APOA1 Rabbit pAb</td>
			<td>Abclone</td>
			<td>A14211</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>TIANGEN</td>
			<td>PA115</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>BLUeye Prestained Protein Ladder</td>
			<td>Sigma-Aldrich</td>
			<td>94964-500UL</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>MP Biomedical</td>
			<td>218072801</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Caveolin-1 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-53564</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>CellMask Orange plasma membrane stain</td>
			<td>Invitrogen</td>
			<td>C10045</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Chemiluminescence</td>
			<td>Amersham Biosciences</td>
			<td>N/A</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Curved operating scissor</td>
			<td>JZ Surgical Instrument</td>
			<td>J21040</td>
			<td>EV isolation</td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>Zhi Ke</td>
			<td>ZK-DST</td>
			<td>EV isolation</td>
		</tr>
		<tr>
			<td>Epoch spectrophotometer</td>
			<td>BioTek</td>
			<td>N/A</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>Eppendorf</td>
			<td>3810X</td>
			<td>EV isolation</td>
		</tr>
		<tr>
			<td>Flotillin-1 antibody</td>
			<td>PTM BIO</td>
			<td>PTM-5369</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Gel imaging system</td>
			<td>Tanon</td>
			<td>4600</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Golgin84</td>
			<td>Novus</td>
			<td>nbp1-83352</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Grids - Formvar/Carbon Coated - Copper 200 mesh</td>
			<td>Polysciences</td>
			<td>24915</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>Heparin Solution</td>
			<td>StemCell&#160;</td>
			<td>7980</td>
			<td>EV isolation</td>
		</tr>
		<tr>
			<td>Liberase Research Grade</td>
			<td>Sigma-Aldrich</td>
			<td>5401127001</td>
			<td>EV isolation</td>
		</tr>
		<tr>
			<td>Microscopic tweezer</td>
			<td>JZ Surgical Instrument</td>
			<td>JD1020</td>
			<td>EV isolation</td>
		</tr>
		<tr>
			<td>NovoCyte flow cytometer</td>
			<td>ACEA</td>
			<td>N/A</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Omni-PAGE Hepes-Tris Gels Hepes 4~20%, 10 wells</td>
			<td>Epizyme</td>
			<td>LK206</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>OSCAR(D-19) antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-34235</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>PBS (2x)</td>
			<td>ZHHC</td>
			<td>PW013</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Sigma-Aldrich</td>
			<td>57-33-0</td>
			<td>Anesthetization</td>
		</tr>
		<tr>
			<td>Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L)</td>
			<td>Jacson</td>
			<td>115-035-003</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)</td>
			<td>Jacson</td>
			<td>111-035-003</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Phosphotungstic acid</td>
			<td>RHAWN</td>
			<td>12501-23-4</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>PKM2(d78a4) xp rabbit&#160; mab&#160;</td>
			<td>Cell Signaling</td>
			<td>4053t</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Polyethylene (PE) film</td>
			<td>Xiang yi</td>
			<td>200150055</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>Polyvinylidene fluoride membranes&#160;</td>
			<td>Roche</td>
			<td>3010040001</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Protease inhibitors</td>
			<td>Roche</td>
			<td>4693132001</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Recombinant anti-PGD antibody</td>
			<td>Abcam</td>
			<td>ab129199</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>RIPA lysis buffer</td>
			<td>Beyotime</td>
			<td>P0013</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>SDS-PAGE loading buffer (5x)</td>
			<td>Cwbio</td>
			<td>CW0027S</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Size beads</td>
			<td>Invitrogen</td>
			<td>F13839</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Tabletop High-Speed Micro Centrifuges</td>
			<td>Hitachi</td>
			<td>CT15E</td>
			<td>EV isolation</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>HITACHI</td>
			<td>H-7650</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>MP Biomedicals</td>
			<td>19472</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Vortex Mixer Genie</td>
			<td>Scientific Industries</td>
			<td>SI0425</td>
			<td>EV isolation</td>
		</tr>
		<tr>
			<td>ZetaView BASIC NTA - Nanoparticle Tracking Video Microscope PMX-120</td>
			<td>Particle Metrix</td>
			<td>N/A</td>
			<td>Nanoparticle tracking analysis</td>
		</tr>
		<tr>
			<td>&#945;-Actinin-4 Rabbit mAb</td>
			<td>Abclone</td>
			<td>A3379</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>&#946;-actin</td>
			<td>Cwbio</td>
			<td>CW0096M</td>
			<td>Western blot</td>
		</tr>
	</tbody>
</table>

isolation and analysis of traceable and functionalized extracellular vesicles from the plasma and solid tissues

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

According to the experimental workflow, EVs can be extracted from the peripheral blood and solid tissues (Figure 1). The maxillary bone of a mouse aged 8 weeks is approximately 0.1 &#177; 0.05 g, and about 300 &#181;L of plasma can be collected from the mouse. Following the protocol steps, 0.3 mg and 3 &#181;g of EVs can be collected, respectively. As analyzed by TEM and NTA, the typical morphological characteristics of EVs are round cup-shaped membrane vesicles with a diameter ranging from ...

Watch this Scientific Journal Video about Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues at JoVE.com

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...

Research

JoVE Journal

Biology

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...