This study was supported by grants from the Natural Science Foundation of China (3157270). We thank Dr. Feng Shao (National Institute of Biological Sciences, China) for providing iBMDM.

1. Isolation of small extracellular vesicles
NOTE: Perform all centrifugation in steps 1.1-1.11 at 4 &#176;C.
<ol>
	<li>Preparation of sEVs-free fetal bovine serum (FBS): Centrifuge FBS overnight at 110,000 &#215; g at 4 &#176;C through an ultracentrifuge (see Table of Materials) to remove endogenous sEVs. Collect the supernatant, filter sterilize it with a 0.2 &#181;m ultrafiltration membrane, and store it at -20 &#176;C.</li>
	<li>Plate about 3 x 107 immortalized bone marrow-derived macrophages (iBMDMs) on a 150 mm culture dish and add 20 mL of DMEM culture medium (see Table of Materials).</li>
	<li>Before collecting sEVs, discard the medium. Wash the cells once with PBS (phosphate-buffered saline; Table of Materials) and replace it with a medium containing 10% sEVs-free FBS.</li>
	<li>According to the needs of the experiment, collect the cell supernatant and transfer it to 50 mL centrifuge tubes.</li>
	<li>Centrifuge the cell supernatant at 300 &#215; g for 10 min to remove the cells, discard the pellet, and transfer the supernatant to new 50 mL centrifuge tubes.</li>
	<li>Centrifuge the supernatant at 2000 &#215; g for 10 min to remove dead cells, discard the pellet, and transfer the supernatant to new high-speed centrifuge tubes (see Table of Materials).</li>
	<li>Centrifuge the supernatant at 10,000 &#215; g for 30 min through a high-speed centrifuge to remove cell debris and microvesicles, discard the pellet, and transfer the supernatant to new ultracentrifuge tubes (see Table of Materials). The volume of the open ultracentrifuge tubes is 38.6 mL; place 35 mL of cell supernatant in each tube.</li>
	<li>Centrifuge the supernatant at 110,000 &#215; g for 70 min in a swinging-bucket rotor centrifuge(see Table of Materials) to obtain crude sEV pellet.</li>
	<li>Discard the supernatant, and wash the sEVs-enriched pellet with 1 mL of PBS. Centrifuge at 110,000 &#215; g for 70 min on a Tabletop ultracentrifuge (see Table of Materials). Discard the supernatant and add 1 mL of PBS to an ultracentrifuge tube.</li>
	<li>Then pipette the precipitate continuously, transfer the 1 mL of PBS to another ultracentrifuge tube, and so on, until all ultracentrifuge tubes are mixed by pipetting.</li>
	<li>Discard the supernatant, and resuspend the pellet in 100 &#181;L of PBS, which is sEVs.</li>
	<li>Measure the total protein of sEVs using the Bicinchoninic Acid (BCA) method, following the steps below (see Table of Materials).
	<ol>
		<li>Preparation of protein standard: Add 1.2 mL of protein preparation solution into a standard protein tube (30 mg of BSA) to fully dissolve it to prepare protein standard solution (25 mg/mL). Then dilute it with PBS to a final concentration of 0.5 mg/mL.</li>
		<li>Add 0 &#181;L, 1 &#181;L, 2 &#181;L, 4 &#181;L, 8 &#181;L, 12 &#181;L, 16 &#181;L, and 20 &#181;L of the protein standard into the 96-well plate, and add PBS to make up 20 &#181;L. At the same time, add 18 &#181;L of HEPES lysis buffer (20 mM HEPES, 50 mM NaCl, 1 mM NaF, 0.5% Triton X-100) and 2 &#181;L of sEVs samples to the sample wells to be tested.</li>
		<li>Prepare the required BCA working solution as per the manufacturer&#39;s instructions, add 200 &#181;L of BCA working solution to each well, and place at room temperature (RT) for 20-30 min.</li>
		<li>Measure the absorbance at 562 nm wavelength with a microplate reader, and calculate the total protein concentration of sEVs according to the standard curve and the dilution factor. 
		NOTE: The sEVs extracted from 40 mL of supernatant by this protocol can be used for subsequent identification of protein markers (western blot). However, sEVs extracted from 200 mL of cell supernatant can be used for both morphological observation (transmission electron microscopy, TEM) and particle size analysis (nanoparticle tracking analysis, NTA). Specifically, for sEVs harvested in step 1.11, resuspend with 100 &#181;L of PBS. Take 20-30 &#181;L for morphology observation (TEM) and resuspend the remaining sample in 1 mL PBS for particle size analysis (NTA). All centrifuges are pre-cooled in advance. For step 1.8, these ultracentrifuge tubes must be strictly balanced and at least three-quarters full. If not, add medium to makeup. For step 1.11, the sEVs can be stored at 4 &#176;C for a short term (12 h); otherwise, they must be stored at -20 or -80 &#176;C to avoid protein degradation that interferes with the extraction of peptides. However, for samples used to observe the morphology and measure particle size, it is only recommended to store at 4 &#176;C for a short term (12 h).</li>
	</ol>
	</li>
</ol>
2. Observation of morphology of sEVs by transmission electron microscopy
<ol>
	<li>Place a drop of fresh sEVs sample (about 20-30 &#181;L) on the parafilm, place the number side of the copper mesh on the sEVs droplet, and let it absorb for 3 min.</li>
	<li>Absorb the excess liquid with filter paper, and then place the copper grid on a drop of distilled water and wash it twice.</li>
	<li>Absorb the excess liquid with filter paper. Then place the copper grid on the 0.5% uranyl acetate droplet for negative staining for 5 s.</li>
	<li>Repeat step 2.2.</li>
	<li>Place the prepared copper mesh on the sample rod and insert it into the sample stage.
	<ol>
		<li>Adjust the magnification to 20,000x-25,000x to find the sample to be tested. Then adjust the magnification to 100,000x and adjust the appropriate position and grayscale to observe under a transmission electron microscope (TEM; Table of Materials). The accelerating voltage for acquiring images is set at 80 kV. 
		NOTE: For step 2.1, if the concentration of sEVs samples is low (&#60;50 &#181;g/&#181;L), the adsorption time can be extended to 5 min or even longer. Alternatively, for step 1.10, a smaller volume of PBS (50 &#181;L) can be used for resuspension. For step 2.3, the negative staining time should not be too long; otherwise, it is difficult to observe the three-dimensional (3D) structure of sEVs.</li>
	</ol>
	</li>
</ol>
3. Measurements of particle size distribution and concentration of sEVs by nanoparticle tracking analysis
<ol>
	<li>For the sEVs harvested in step 1.11, dilute the solution to 1 mL for nanoparticle tracking analysis (NTA). First, clean the instrument with distilled water until there are no impurities in the field of vision. Then use a 1 mL sterile syringe to push the sample slowly (injection volume: 0.5-1 mL).</li>
	<li>Analyze the samples through supporting software (see Table of Materials). Specifically, click Start Camera and adjust the Camera level to an appropriate size (generally an intensity of 14-16 units), then select the measurement method, Standard Measurement (3 or 5 times), and click Run to collect the particles.</li>
	<li>After collecting the particles, the computer can observe the moving sEVs particles. At the same time, set the detection threshold (generally 5) to analyze the particle size distribution so that the final counted particles are all moving sEVs rather than the background. After analyzing the particles, save and export the analysis results.</li>
	<li>After use, wash the instrument with distilled water until there are no obvious particles in the field of vision, and turn off the laser. 
	NOTE: Unlike sEVs samples for TEM, sEVs samples for NTA should not be too concentrated and require larger sample volumes (at least 1 mL). Additionally, alternative methods can be used to analyze the size distribution of sEVs, such as flow cytometry and tunable resistive pulse sensing (TRPS), etc. The recommended number of particles/frames for NTA measurements (NanoSight LM10) is 40.</li>
</ol>
4. Detection of protein markers of sEVs by western blot
NOTE: According to the Minimal information for studies of extracellular vesicles (MISEV) 2018 guidelines17,18, 5 categories of proteins are recommended for the characterization of sEVs. Evaluate at least one protein marker of each category 1 to 4 for sEVs preparation.
<ol>
	<li>For the sEVs harvested in step 1.9, carefully pipette the supernatant, and add 15 &#181;L of HEPES lysis buffer for 5 min. Then add an equal amount of loading buffer and cook in boiling water for 15 min.</li>
	<li>In this protocol, the loading of sEVs&#39; protein was 8-10 &#181;g. Electrophorese the proteins in 10% gels at a constant voltage of 80 V (about 30 min).
	<ol>
		<li>When the sample enters the separating gel, adjust the voltage to 120 V (about 45 min). After the sample is 2-3 cm away from the bottom of the glass plate, disconnect the power supply and cut off the sample strip for electroporation. The composition of electrophoresis solution is described in sub-step 4.2.1.1.
		<ol>
			<li>To prepare 5x electrophoresis solution, weigh 15.1 g, 5 g, and 94 g of Tris, SDS, and glycine, respectively, and dilute to 1 L with deionized water. When it is used, it needs to be diluted 5 times with water.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Assemble the electroporation device and put in sufficient electroporation solution (about 1 L), and electroporate the protein on the nitrocellulose (NC) membrane with a constant current of 90 mA on ice for 3.5 h. 
	NOTE: To prepare 10x electroporation solution, weigh 30.25 g and 144.1 g of Tris and glycine, respectively, and dilute to 1 L with deionized water. When it is used, it needs to be prepared at a ratio of 7:2:1 of deionized water: methanol: electroporation solution.</li>
	<li>After electroporation, place the NC membrane in blocking solution (2.5 g of skimmed milk powder in 50 mL of Tris-buffered saline with 0.1% Tween 20 (TBST) for 1-2 h at RT or overnight at 4 &#176;C. The composition of TBST is described in sub-step 4.4.1.
	<ol>
		<li>To prepare 10x TBST, weigh 60.56 g and 175.32 g of Tris and NaCl, respectively, and then add 10 mL of Tween-20 and 34 mL of concentrated hydrochloric acid. Adjust pH = 7.6 with concentrated hydrochloric acid and makeup to 2 L with deionized water. When it is used, it needs to be diluted ten times with water.</li>
	</ol>
	</li>
	<li>Identify the protein markers by three sEVs&#39; markers (cluster of differentiation 9 [CD9]; &#946;-actin; tumor susceptibility gene [TSG101]) and one non- sEVs&#39; marker (glucose-regulated protein 94 [GRP94]).
	<ol>
		<li>Pipette 2 &#181;L of the above antibodies and dilute at 1:500, then incubate the NC membranes at RT for 3 h or overnight at 4 &#176;C. The diluent is the blocking solution in step 4.4.</li>
		<li>After incubation with the primary antibody, wash 3 times with 1x TBST on a shaker for 10 min each time.</li>
	</ol>
	</li>
	<li>Place the NC membrane in the diluted secondary antibody (1:500) and shake slowly on a shaker at RT for 45-50 min. Wash it 3 times with 1x TBST on a shaker for 10 min each time.</li>
	<li>Mix the chemiluminescent substrates A and B (see&#160;Table of Materials) at 1:1, add the mixed reagent to the NC membrane, and incubate at RT for 5 min.</li>
	<li>Image the NC membrane using a blot imager (see Table of Materials)
	<ol>
		<li>Open the Image Lab software, and select New Experimental Protocol.</li>
		<li>Select the application Blotting-colorimetric, cancel Highlight, click Run Experimental Protocol, acquire the marker, and save it.</li>
		<li>Then re-select the application program Blotting-chemi, and set the Total number of images and Exposure time to 30 s and 300 s, respectively.</li>
		<li>Click Run Experimental Protocol, acquire the images, and save it. Finally, remove the NC membrane and turn off the computer. 
		NOTE: For step 4.6, the incubation time of the secondary antibody should not exceed 50 min; otherwise, the background will be too dark.</li>
	</ol>
	</li>
</ol>
5. Extraction of sEVs&#39; peptides
<ol>
	<li>Wash the sEVs twice by ultracentrifugation at 110,000 &#215; g at 4 &#176;C for 70 min, and add 500 &#181;L of PBS (see Table of Materials) to resuspend them, and add 5 &#181;L of 100x protease inhibitor (see Table of Materials).</li>
	<li>Place the sample for ultrasonic crushing (see Table of Materials). Settings for ultrasonic homogenization are as follows: power: 40 W, total time: 10 min, ultrasonic time: 3 s, and interval time: 5 s.</li>
	<li>Centrifuge the crushed mixture at 13,700 &#215; g for 15 min at 4 &#176;C.</li>
	<li>After centrifugation, add dithiothreitol (DTT; Table of Materials) to the supernatant to a final concentration of 50 mmol/L, and incubate in a water bath at 56 &#176;C for 30 min.</li>
	<li>Subsequently, add 50 &#181;L of iodoacetamide (IAA; Table of Materials) to the supernatant at a concentration of 1 mol/L and leave it to react at RT for 20 min in the dark.</li>
	<li>Centrifuge the mixture at 13,700 &#215; g at 4 &#176;C for 15 min, and collect the supernatant, the crude peptides extract. Store it at -20 &#176;C for later use.</li>
	<li>Ultrafilter the obtained peptides crude extract with a 10 kDa ultrafiltration tubes19&#160;(see Table of Materials) to remove proteins, and centrifuge at 13,700 &#215; g at 4 &#176;C for 1 h. Collect the effluent and dry it in a vacuum centrifugal concentrator (see Table of Materials) at 45 &#176;C.</li>
	<li>Desalt the dried samples following steps 5.8.1-5.8.8. Use mass spectrometry-grade pure water as a solvent for the reagents.
	<ol>
		<li>Add 100 &#181;L of 0.1% trifluoroacetic acid (TFA) to each sample to completely dissolve the dried peptides.</li>
		<li>Add 100 &#181;L of 100% acetonitrile (ACN) to each desalting column, centrifuge at 400 &#215; g for 3 min at RT, and discard the effluent. Then add 100 &#181;L of 50% ACN, centrifuge at 400 &#215; g for 3 min, and discard the effluent.</li>
		<li>Add 100 &#181;L of 0.1% TFA to each desalting column, centrifuge at 400 &#215; g for 3 min, and discard the effluent.</li>
		<li>Repeat step 5.8.3.</li>
		<li>Pipette the peptides dissolved in step 5.8.1 into the desalting column, centrifuge at 400 &#215; g for 3 min, and recover the effluent. Repeat the sample loading step to let the peptide sample adsorb into the desalting column.</li>
		<li>Repeat step 5.8.3 twice.</li>
		<li>Add 50 &#181;L of 50% ACN (containing 0.1% TFA) to the desalting column, centrifuge at 400 &#215; g for 3 min, and collect the effluent (peptides) in a new mass spectrometry-grade centrifuge tube.</li>
	</ol>
	</li>
</ol>
6. Drying the desalted peptides
<ol>
	<li>Dry the desalted peptides in a vacuum centrifugal concentrator (Settings: V-AQ mode, temperature: 45 &#176;C, and time: 50 min ) and use them for subsequent mass spectrometry analysis. 
	NOTE: The extraction process of sEVs&#39; peptides should be performed on ice as much as possible to avoid protein degradation. All reagents used were prepared with mass spectrometry-grade water to avoid plastic contamination. For step 5.8, desalting results in the inevitable loss of the peptide samples. This loss can be reduced by repeating steps 5.8.5 and 5.8.7.</li>
</ol>
7. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis
NOTE: Analyze the peptides&#39; sample by liquid chromatography-tandem mass spectrometry (LC-MS/MS), specifically the Orbitrap Q Exactive HF-X mass spectrometer connected with an EASY-nLC 1000 nano-high-performance LC system (see Table of Materials).
<ol>
	<li>Separate the samples on a C18 analytical column (1.9 &#181;m of grain diameter, 15 cm length x 150 &#181;m inner diameter) at a flow rate of 60 nL/min with a 65 min gradient: 4%-15% for 4 min, 15%-28 % for 28 min, 28%-40% for 10 min, 40%-69% for 10 min, 95% constant for 7 min, 95% decreased to 6% for 1 min, and constant for 5 min (solvent A, water containing 0.1% formic acid [FA]; solvent B, 80% acetonitrile containing 0.1% FA, v/v).</li>
	<li>Ionize the eluted peptides in a nano-electrospray ionization (nano-ESI) sprayer at 320 &#176;C capillary temperature and spray voltage of 2.2 kV.</li>
	<li>Collect the mass spectral data using data-dependent acquisition mode with a resolving power of 120,000 for full-scan mode and 15,000 at MS/MS mode.
	<ol>
		<li>Perform full scans in the orbitrap from scan range 250 m/z to 1800 m/z and isolation window with 1.6 m/z.</li>
		<li>Select the top 20 intense ions for higher-energy collisional dissociation (HCD) fragmentation with a normalized collision energy of 29% and measure in the ion separator. 
		NOTE: Typical mass spectrometric conditions were as follows: Automatic gain control (AGC) targets were 3 x 106 ions for full scans and 2 x 105 for MS/MS scans; the maximum injection time was 80 ms for full scans and 19 ms for MS/MS scans; and dynamic exclusion was used for 13 s.</li>
	</ol>
	</li>
</ol>

Isolating Small Extracellular Vesicles and Extracting Their Peptidome

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The authors declare that they have no competing financial interests.

When investigating the function of sEVs, it is imperative to attain high-purity sEVs from complex biological samples to avoid any potential contaminations. A variety of methods for sEVs isolation have been developed13, and among these methods, differential ultracentrifugation-based methods have shown relatively high purity of sEVs. In this study, 200 mL of cell supernatant was collected for 6 h, and about 200-300 &#181;g of sEVs were obtained by differential ultracentrifugation. However, it should...

Small extracellular vesicles (sEVs) with a diameter of less than 200 nm are present in almost all types of body fluids and secreted by all kinds of cells, including urine, sweat, tears, cerebrospinal fluid, and amniotic fluid1. Initially, sEVs were considered as receptacles for disposing of cellular waste, which led to minimal research in the subsequent decade2. Recently, increasing evidence indicates that sEVs contain specific proteins, lipids, nucleic acids, and other metabolites. These molecules are transported to target cells3, contributing to intercellular communication, through which they participate in various biological processes, such as tissue repair, angiogenesis, immunity4 and inflammation5,6, tumor development and metastasis7,8,9, etc.
To facilitate the study of sEVs, it is imperative to isolate sEVs from complex samples. Different sEVs isolation methods have been developed based on the physical and chemical properties of sEVs, such as their density, particle size, and surface marker proteins. These techniques include ultracentrifugation-based methods, particle size-based methods, immunoaffinity capture-based methods, sEVs precipitation-based methods, and microfluidics-based methods10,11,12. Among these techniques, the ultracentrifugation-based method is widely recognized as the gold standard for sEVs isolation and is the most commonly used technique13.
An increasing amount of evidence suggests the presence of a multitude of undiscovered biologically active peptides in the peptidomes of various organisms. These peptides significantly contribute to numerous physiological processes by regulating growth, development, stress response14,15, and signal transduction16. The objective of sEVs&#39; peptidome is to uncover the peptides carried by these sEVs and provide clues to their biological functions. Here, we present a protocol of isolating sEVs through differential ultracentrifugation, followed by extraction of peptides from these sEVs for further analysis of their peptidome.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Beyotime Technology</td>
			<td>P0012</td>
			<td></td>
		</tr>
		<tr>
			<td>CD9</td>
			<td>Beyotime Technology</td>
			<td>AF1192</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugal filter tube</td>
			<td>Millipore</td>
			<td>UFC5010BK</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge bottles polypropylene</td>
			<td>Beckman Coulter</td>
			<td>357003</td>
			<td>High-speed centrifuge</td>
		</tr>
		<tr>
			<td>Chemiluminescent substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>34580</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Solarbio</td>
			<td>D8220</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>DMEM culture medium</td>
			<td>Cell World</td>
			<td>N?A</td>
			<td></td>
		</tr>
		<tr>
			<td>GRP94</td>
			<td>Cell Signaling Technology</td>
			<td>20292</td>
			<td></td>
		</tr>
		<tr>
			<td>High-speed centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Avanti JXN-26</td>
			<td>Centrifuge rotor (JA-25.50)</td>
		</tr>
		<tr>
			<td>Immortalized bone marrow-derived macrophages (iBMDM)</td>
			<td>National Institute of Biological Sciences, China</td>
			<td></td>
			<td>Provided by Dr. Feng Shao (National Institute of Biological Sciences, China)</td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma</td>
			<td>l1149</td>
			<td>5 g</td>
		</tr>
		<tr>
			<td>Microfuge tube polypropylene</td>
			<td>Beckman Coulter</td>
			<td>357448</td>
			<td>1.5 mL, Tabletop ultracentrifuge&#160;</td>
		</tr>
		<tr>
			<td>nano-high-performance LC system</td>
			<td>Thermo Fisher Scientific</td>
			<td>EASY-nLC 1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoparticle tracking analysis&#160;</td>
			<td>Malvern Panalytical</td>
			<td>NanoSight LM10</td>
			<td>NanoSight NTA3.4</td>
		</tr>
		<tr>
			<td>Orbitrap Q Exactive HF-X mass spectrometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Solarbio</td>
			<td>P1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyallomer centrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td>326823</td>
			<td>Ultracentrifuge</td>
		</tr>
		<tr>
			<td>Protease inhibitor</td>
			<td>Bimake</td>
			<td>B14002</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac vacuum concentrator</td>
			<td>Eppendorf</td>
			<td>Concentrator plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima MAX-XP</td>
			<td>Ultracentrifuge rotor (TLA 55)</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>HITACHI</td>
			<td>H-7650B</td>
			<td></td>
		</tr>
		<tr>
			<td>TSG101</td>
			<td>Sigma</td>
			<td>AF8258</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima XPN-100</td>
			<td>Ultracentrifuge rotor (SW32 Ti)</td>
		</tr>
		<tr>
			<td>Ultrasonic cell disruptor</td>
			<td>Scientz</td>
			<td>SCIENTZ-IID</td>
			<td></td>
		</tr>
		<tr>
			<td>Western Blot imager</td>
			<td>Bio-Rad</td>
			<td>ChemiDocXRs</td>
			<td>Image lab 4.0 (beta 7)</td>
		</tr>
		<tr>
			<td>&#946;-actin</td>
			<td>Sigma</td>
			<td>A3853</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of peptides of small extracellular vesicles from bone marrow-derived macrophages

Small extracellular vesicles (sEVs) are typically secreted by the exocytosis of multivesicular bodies (MVBs). These nanovesicles with a diameter of &lt;200 nm are present in various body fluids. These sEVs regulate various biological processes such as gene transcription and translation, cell proliferation and survival, immunity and inflammation through their cargos, such as proteins, DNA, RNA, and metabolites. Currently, various techniques have been developed for sEVs isolation. Among them, the ultracentrifugation-based method is considered the gold standard and is widely used for sEVs isolation. The peptides are naturally biomacromolecules with less than 50 amino acids in length. These peptides participate in a variety of biological processes with biological activity, such as hormones, neurotransmitters, and cell growth factors. The peptidome is intended to systematically analyze endogenous peptides in specific biological samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Here, we introduced a protocol to isolate sEVs by differential ultracentrifugation and extracted peptidome for identification by LC-MS/MS. This method identified hundreds of sEVs-derived peptides from bone marrow-derived macrophages.

For the sEVs isolated by differential ultracentrifugation (Figure 1), we evaluated their morphology, particle size distribution, and protein markers according to the International Society for Extracellular Vesicles (ISEV)17.
First, the morphology of sEVs was observed by TEM, showing a typical cup-like structure (Figure 2A). NTA showed that isolated sEVs were mostly concentrated at 136 nm (F...

Watch this Scientific Journal Video about Peptidome Extraction from Small Extracellular Vesicles Isolated from Bone Marrow-Derived Macrophages at JoVE.com

Author Spotlight: Peptidome Extraction from Small Extracellular Vesicles Isolated from Bone Marrow-Derived Macrophages 

This protocol describes a procedure to isolate small extracellular vesicles from macrophages by differential ultracentrifugation and extract the peptidome for identification by mass spectrometry.

Identification of Peptides of Small Extracellular Vesicles from Bone Marrow-Derived Macrophages

Small extracellular vesicles (sEVs) are typically secreted by the exocytosis of multivesicular bodies (MVBs). These nanovesicles with a diameter of ...

identification-peptides-small-extracellular-vesicles-from-bone-marrow

Research

JoVE Journal

Biochemistry

1.9K Views.  Anhui Medical University. This protocol describes a procedure to isolate small extracellular vesicles from macrophages by differential ultracentrifugation and extract the peptidome for identification by mass spectrometry.

Video: Author Spotlight: Peptidome Extraction from Small Extracellular Vesicles Isolated from Bone Marrow-Derived Macrophages 

Extraction of Extracellular Vesicles from Whole Tissue

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or prognostic biomarker assays, and likely serve as important players in the progression of a vast spectrum of diseases. Current research is focused on the characterization of vesicles secreted from multiple cell and tissue types in order to better understand the role of EVs in the pathogenesis of conditions including neurodegeneration, inflammation, and cancer. However, globally consistent and reproducible techniques to isolate and purify vesicles remain in progress. Moreover, methods for extraction of EVs from solid tissue ex vivo are scarcely described. Here, we provide a detailed protocol for extracting small EVs of interest from whole fresh or frozen tissues, including brain and tumor specimens, for further characterization. We demonstrate the adaptability of this method for multiple downstream analyses, including electron microscopy and immunophenotypic characterization of vesicles, as well as quantitative mass spectrometry of EV proteins.

Here, we provide a detailed protocol to isolate small extracellular vesicles (EVs) from whole tissues, including brain and tumor specimens. This method offers a reproducible technique to extract EVs from solid tissue for further downstream analyses.

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or ...

Cancer Research

Enhancing EV Isolation for Precise Biofluid Analysis

Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of cell, they provide a real-time snapshot of host cells and contain a wealth of molecular information, including proteins, in particular those with post-translational modifications (PTMs) such as phosphorylation, as the main player of cellular functions and disease onset and progression. However, the isolation of EVs from biofluids remains challenging due to low yields and impurities from current EV isolation methods, making the downstream analysis of EV cargo, such as EV phosphoproteins, difficult. Here, we describe a rapid and effective EV isolation method based on functionalized magnetic beads for EV isolation from biofluids such as human urine and downstream proteomics and phosphoproteomics analysis following EV isolation. The protocol enabled a high recovery yield of urinary EVs and sensitive profiles of EV proteome and phosphoproteome. Furthermore, the versatility of this protocol and relevant technical considerations are also addressed here.

Author Spotlight: Advancing EVtrap for High-Throughput Proteomics in Disease Biomarker Discovery 

The present protocol provides detailed descriptions for the efficient isolation of urinary extracellular vesicles utilizing functionalized magnetic beads. Moreover, it encompasses subsequent analyses, including western blotting, proteomics, and phosphoproteomics.

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of ...

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a variety of particles. Normally, BMDMs are restricted from the central nervous system (CNS), but following an injury, they can readily infiltrate. Once within the injured CNS tissue, BMDMs are the primary cell type responsible for the clearance of injury-derived cellular debris, including large quantities of lipid rich myelin debris. The neuropathological ramifications of BMDM infiltration and myelin debris phagocytosis within the CNS are complex and not well understood. The protocols described here, allow for the direct in vitro study of BMDMs in the context of CNS injury. We cover murine BMDM isolation and culture, myelin debris preparation, and assays to assess BMDM myelin debris phagocytosis. These techniques produce robust quantifiable results without the need for significant specialized equipment or materials, yet can be easily customized to meet the needs of researchers.

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

We present methods to assess the phagocytic capacity of primary murine bone marrow-derived macrophages using fluorescently labeled myelin debris and intracellular lipid droplet staining.

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a ...

Neuroscience

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven&#160;OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 &#181;m sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their ...

Bioengineering

Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident immune cells in the brain, and sometimes the infiltrating bone marrow-derived macrophages (BMDMs), regulate the inflammatory profile of their microenvironment in the CNS. It is now accepted that the extracellular vesicle (EV) populations from immune cells act as immune mediators. Thus, their collection and isolation are important to identify their contents but also evaluate their biological effects on recipient cells. The present data highlight chronological requirements for EV isolation from microglia cells or blood macrophages including the ultracentrifugation and size-exclusion chromatography (SEC) steps. A non-targeted proteomic analysis permitted the validation of protein signatures as EV markers and characterized the biologically active EV contents. Microglia-derived EVs were also functionally used on primary culture of neurons to assess their importance as immune mediators in the neurite outgrowth. The results showed that microglia-derived EVs contribute to facilitate the neurite outgrowth in vitro. In parallel, blood macrophage-derived EVs were functionally used as immune mediators in spheroid cultures of C6 glioma cells, the results showing that these EVs control the glioma cell invasion in vitro. This report highlights the possibility to evaluate the EV-mediated immune cell functions but also understand the molecular bases of such a communication. This deciphering could promote the use of natural vesicles and/or the in vitro preparation of therapeutic vesicles in order to mimic immune properties in the microenvironment of CNS pathologies.

The present report highlights chronological requirements for extracellular vesicle (EV) isolation from microglia or blood macrophages. Microglia-derived EVs were evaluated as regulators of the neurite outgrowth while blood macrophage-derived EVs were studied in the control of C6 glioma cell invasion in in vitro assays. The goal is to better understand these EV functions as immune mediators in specific microenvironments.

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident ...

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 regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

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

Biology

Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem Cells and their Derived Extracellular Vesicles

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of pluripotent stem cells, are favored for their instant availability and proven safety. It has been reported that transplantation of BM-MSCs is of great benefit to repairing injured tissues in various diseases, which might be related to modulating the immune and inflammatory responses via paracrine mechanisms. Extracellular vesicles (EVs), featuring a double-layer lipid membrane structure, are considered to be the main mediators of the paracrine effects of stem cells. Recognized for their crucial roles in cell communication and epigenetic regulation, EVs have already been applied in vivo for immunotherapy. However, similar to its maternal cells, most of the studies on the efficacy of transplantation of EVs still remain at the level of small animals, which is not enough to provide essential evidence for clinical translation. Here, we use density-gradient centrifugation to isolate bone marrow cells (BMC) from porcine bone marrow at first, and get porcine BM-MSCs (pBM-MSCs) by cell culture subsequently, identified by the results of observation under the microscope, induced differentiation assay, and flow cytometry. Furthermore, we isolate EVs derived from pBM-MSCs in cell supernatant by ultracentrifugation, proved by the techniques of transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting successfully. Overall, pBM-MSCs and their derived EVs can be isolated and identified effectively by the following protocols, which might be widely used in pre-clinical studies on the transplantation efficacy of BM-MSCs and their derived EVs.

This article elaborates a method to isolate and identify porcine bone marrow mesenchymal stem cells (pBM-MSCs) and extracellular vesicles (EVs) derived from them, providing a methodological basis for the pre-clinical evaluation of transplantation efficacy of BM-MSCs and their derived EVs.

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of ...

Medicine

Efficient Isolation of Brain sEVs from Minimal Tissue for Biomarker Discovery

Harnessing the Power of MicroRNA Cargoes in Small Extracellular Vesicles Released from Fresh-Frozen Human Brain Sections

Small extracellular vesicles (sEVs) are crucial mediators of cell-cell communication, transporting diverse cargoes like proteins, lipids, and nucleic acids (microRNA, mRNA, DNA). The microRNA sEV cargo has potential utility as a powerful non-invasive disease biomarker due to sEV&#39;s ability to traverse biological barriers (e.g., blood-brain barrier) and become accessible through various body fluids. Despite numerous studies on sEV biomarkers in body fluids, identifying tissue or cell-specific sEV subpopulations remains challenging, particularly from the brain. Our study addresses this challenge by adapting existing methods to isolate sEVs from minimal amounts of frozen human brain sections using size exclusion chromatography (SEC).
After ethical approval, approximately 250 &#181;g of fresh-frozen human brain tissue (obtained from Manchester Brain Bank [UK]) was sliced from the 3 donor tissues and incubated in collagenase type 3/Hibernate-E solution, with intermediate agitation, followed by serial centrifugation and filtration steps. Then, sEVs were isolated using the SEC method and characterized by following MISEV guidelines. Before isolating RNA from within these sEVs, the solution was treated with Proteinase-K and RNase-A to remove any non-sEV extracellular RNA. The RNA quantity and quality were checked and processed further for qPCR and small RNA sequencing experiments.
The presence of sEVs was confirmed through fluorescence nanoparticle tracking analysis (fNTA) and western blot for surface markers (CD9, CD63, CD81). Size distribution (50-200 nm) was confirmed by NTA and electron microscopy. The total RNA concentration within lysed sEVs ranged from 3-9 ng/&#181;L and was used for successful quantification by qPCR for selected candidate microRNAs. Small RNA sequencing on MiSeq provided high-quality data (Q &#62;32) with 1.4-5 million reads per sample.
This method enables efficient isolation and characterization of sEVs from minimal brain tissue volumes, facilitating non-invasive biomarker research and holds promise for equitable disease biomarker studies, offering insights into neurodegenerative diseases and potentially other disorders.

Here, we establish a protocol using minimal amounts of fresh-frozen brain tissue sections and an accessible high-speed centrifugation method coupled with size exclusion chromatography to obtain small extracellular vesicles as sources for microRNA (miRNA) biomarkers for neurological disorders.

Small extracellular vesicles (sEVs) are crucial mediators of cell-cell communication, transporting diverse cargoes like proteins, lipids, and nucleic ...

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and first-generation particle analysis to comprehensive multi-parameter measurements such as nano-flow cytometry (nFCM). nFCM is a form of flow cytometry that utilizes instrumentation specifically designed for nano-particle analysis, allowing for thousands of EVs to be characterized per minute both with and without the use of staining techniques. High resolution side scatter (SS) detection allows for size and concentration to be determined for all biological particles larger than 45 nm, while simultaneous fluorescence (FL) detection identifies the presence of labeled markers and targets of interest. Labeled subpopulations can then be described in quantitative units of particles/mL or as a percentage of the total particles identified by side scatter.
Here, EVs derived from conditioned cell culture media (CCM) are labeled with both a lipid dye, to identify particles with a membrane, and antibodies specific for CD9, CD63, and CD81 as common EV markers. Measurements of comparison material, a concentration standard and a size standard of silica nanospheres, as well as labeled sample material are analyzed in a 1-minute analysis. The software is then used to measure the concentration and size distribution profile of all particles, independent of labeling, before determining the particles that are positive for each of the labels.
Simultaneous SS and FL detection can be utilized flexibly with many different EV sources and labeling targets, both external and internal, describing EV samples in a comprehensive and quantitative manner.

The latest generation of EV characterization tools are capable of single EV analysis across multiple parameters simultaneously. Nano-flow cytometry measures all biological particles larger than 45 nm without labeling and identifies specific characteristics of subpopulations by a variety of fluorescent labeling techniques.

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and ...

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs&#39; size was 30-200 nm, with a particle concentration of 7.75 &#215; 1010 particles/mL and a protein concentration of 80 &#181;g/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells&#39; small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this ...