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>
	<li>Abels, E. R., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+extracellular+vesicles:+biogenesis,+RNA+cargo+selection,+content,+release,+and+uptake.">Introduction to extracellular vesicles: biogenesis, RNA cargo selection, content, release, and uptake.</a> Cellular and Molecular Neurobiology. 36 (3), 301-312 (2016).</li><li>Mathieu, M., Martin-Jaular, L., Lavieu, G., Th&#233;ry, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificities+of+secretion+and+uptake+of+exosomes+and+other+extracellular+vesicles+for+cell-to-cell+communication.">Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication.</a> Nature Cell Biology. 21 (1), 9-17 (2019).</li><li>Van Niel, G., D'Angelo, G., Raposo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shedding+light+on+the+cell+biology+of+extracellular+vesicles.">Shedding light on the cell biology of extracellular vesicles.</a> Nature Reviews Molecular Cell Biology. 19 (4), 213-228 (2018).</li><li>Witwer, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+sample+collection,+isolation+and+analysis+methods+in+extracellular+vesicle+research.">Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.</a> Journal of Extracellular Vesicles. 2 (1), 20360 (2013).</li><li>Keshtkar, S., Azarpira, N., Ghahremani, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell-derived+extracellular+vesicles:+novel+frontiers+in+regenerative+medicine.">Mesenchymal stem cell-derived extracellular vesicles: novel frontiers in regenerative medicine.</a> Stem Cell Research &amp; Therapy. 9 (1), 63 (2018).</li><li>Thietart, S., Rautou, P. 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G., Wurdinger, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-educated+pletelets.">Tumor-educated pletelets.</a> Blood. 133 (22), 2359-2364 (2019).</li><li>Schwager, S. C., Reinhart-King, C. 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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 ...

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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 ...

This protocol provides a feasible way to isolate and collect the right extracellular vesicles from most blood and solid tissues, especially analyze their source and the protein contents, which helps further functional experiments. Besides high yield and low contamination, the main advantage of this protocol is analysis of surface antigens and protein cargoes of EVs which is useful for physiological and pathological studies. It facilitates the diagnosis of cancers of diseases, such as inflammatory diseases and osteoporosis through the detection of surface markers of parental cells of extracellular vesicles with a flow cytometer.The quantity of tissue extracellular vesicle is critical for the following experiments. Make sure the concentration of extracellular vesicle is not high. Be patient to perform the dilution.To begin, prepare the maxillary bone samples by isolating the maxillary bone with ophthalmic tweezers and scissors and wash them with PBS to get rid of soft tissues with tweezers. Then put the maxillary bone into 1.5 milliliter centrifuge tubes and cut the bone into small pieces of one millimeter in diameter with scissors. Add a certain amount of Liberase to cover the tissue and incubate for 30 minutes at 37 degrees Celsius.Next, centrifuge the sample at 800 G for 10 minutes at four degrees Celsius and carefully transfer the supernatant with a pipette to a new and clean 1.5 milliliter centrifuge tubes. After centrifuging the prepared plasma and bone samples for 15 minutes at 2, 500 G and four degree Celsius to remove large cell debris and remaining platelets, carefully transfer the supernatants to new and clean 1.5 milliliter centrifuge tubes and centrifuge at 16, 800 G for 30 minutes at four degrees Celsius. Next, discard the supernatant and resuspend the pellets in each tube with one milliliter of PBS.After centrifuging and discarding the supernatant as demonstrated previously, again, resuspend the pellets in each tube with 50 microliters of PBS and store these samples at four degrees Celsius for less than 24 hours or preferably use them immediately for the analyses. Resuspend samples with 500 microliters of PBS and transfer 50 microliters into a new and clean 1.5 milliliter centrifuge tube as a blank control labeled as tube A and another 50 microliters into another clean 1.5 milliliter centrifuge tube as a simple staining tube for FITC, labeled as tube B.Add 0.5 microliters of membrane dye to the primary tube for membrane staining while incubating for five minutes at room temperature. After centrifuging the primary tube for 30 minutes at 16, 800 G and discarding the supernatant, resuspend the pellets with 200 microliters of PBS.Divide each 50 microliter sample into four 1.5 milliliter centrifuge tubes for simple staining tubes for PE labeled as tube C, surface marker staining labeled as tube D, and secondary antibody-only controls labeled as tube E.Add the primary antibody of osteoclast associated receptor and bone extracellular vesicle samples as well as CD-18 antibody and plasma extracellular vesicle samples to tube B and tube D separately, incubating for one hour at four degrees Celsius. Centrifuge the tubes and discard the supernatant as demonstrated previously and resuspend the pellets with 500 microliters of PBS. Again, centrifuge for 30 minutes at 16, 800 G and four degree Celsius to remove the extra primary antibodies.After discarding the supernatant resuspend the pellets with 50 microliters of PBS, followed by adding FITC conjugated secondary antibodies respectively in tube E.Incubate all the tubes for one hour at four degree Celsius in the dark. Dilute one drop of 0.2, 0.5, and one micrometer sized bead suspension respectively into one milliliter of PBS. Then run each size of beads to make sure of the gate chosen for extracellular vesicles and set the threshold of the flow cytometer to search for the beads and extracellular vesicle population using a suitable forward inside scatter.Set the terminal condition as calculating 100, 000 membrane dyed particles and analyze the sample via a flow cytometer as described in the manuscript. Resuspend the pellets in 50 microliters of RIPA-lysis buffer and incubate for 30 minutes on ice. To quantify the protein concentrations of all samples in the 96 well microplate by the BCA protein assay, dilute the two milligrams per milliliters of BSA with 0.9%of normal saline into 0.5 milligrams per milliliters.At 0.5 milligrams per milliliter of BSA and 0.9%of normal saline into three duplicated wells with certain volumes. Then drop two microliters of the samples and add 18 microliters of normal saline into three duplicated wells separately. After preparing a working solution by mixing BCA reagent A with reagent B, add 200 microliters of the working solution to each well and shake gently for 30 seconds.Then incubate the 96 well microplate for 20 to 25 minutes at 37 degrees Celsius and measure the optical density at 596 nanometers with the spectrophotometer. Next, export the data, draw a standard curve, and calculate the protein concentration of the samples. According to the results, dilute the samples to one microgram per microliter with 0.9%of normal saline and five times the concentration of SDS-PAGE loading buffer and seal the tubes with film tightly and heat for five minutes at 100 degrees Celsius.Load the samples in the protein ladder into a gradient concentration of four to 20%HEPy's tris gel. Run the gel in the running buffer at 80 volts until the proteins form a line. Then switch to 120 volts for one hour until the loading dye is at the bottom of the gel.Transfer the gel to the polyvinylidene fluoride membrane pre incubated in methyl alcohol for 20 seconds using a wet transfer system to transfer for one hour at 200 milliamperes. Prepare 5%BSA blocking buffer by adding 2.5 grams of BSA and 50 milliliters of TBST into a 50 milliliter centrifuge tube. Block the membranes in this buffer for two hours at room temperature with agitation.Incubate the membranes with specific primary antibodies diluted with TBST into proper concentration overnight at four degrees Celsius. Wash the membranes in the washing buffer four times and incubate the membranes with suitable secondary antibodies for one hour at room temperature with agitation. After washing the membranes in the washing buffer four times, image the membranes using the chemiluminescence kit in a gel imaging system.The transmission electron microscopy and nanoparticle tracking analysis revealed that the typical morphological characteristics of extracellular vesicles were round and cup shaped with a diameter ranging from 500 to 300 nanometers. Flow cytometry analysis shows the percentages of specific membrane markers expressed on extracellular vesicles that implies their origin from parent cells. Western blot analysis shows the expression of PGD and PKM2 respectively as a representative of protein content and plasma extracellular vesicles and bone extracellular vesicles, indicating the metabolic status.Negative expression of Golgin-84 in extracellular vesicles was detected with the presence of mitofilin, Alpha-actinin-4, Flotillin-1, Caveolin-1, and Beta-actin, along with vesicles marker CD9, while CD81 expression is low or absent with a lack of APO-A-1 existence in the plasma extracellular vesicles. Researchers, so keeping in mind that step 2.2, the preparation and the digestion of the tissue samples should be done sufficiently. Otherwise, the number of extracellular vesicles extracted from the tissues is limited.The marker labeled fluorescent stain of the extracellular vesicles can be injected into the mouth to treat the phase that linked to potential function.

isolation-analysis-traceable-functionalized-extracellular-vesicles

Research

JoVE Journal

Biology

1.8K vistas.  The Fourth Military Medical University. Este protocolo proporciona una forma factible de aislar y recolectar las vesículas extracelulares correctas de la mayoría de los tejidos sanguíneos y sólidos, especialmente analizar su fuente y el contenido de proteínas, lo que ayuda a realizar experimentos funcionales. Además del alto rendimiento y la baja contaminación, la principal ventaja de este protocolo es el análisis de antígenos superficiales y cargas de proteínas de EV, que es útil para estudios fisiológicos y patológic...