The presented work was supported by the Ministe&#768;re de L&#8217;Education Nationale, de L&#8217;Enseignement Supe&#769;rieur et de la Recherche and INSERM. We gratefully acknowledge the BICeL- Campus Scientific City Facility for access to instruments and technical advices. We gratefully acknowledge Jean-Pascal Gimeno, Soulaimane Aboulouard and Isabelle Fournier for the Mass spectrometry assistance. We gratefully acknowledge Tanina Arab, Christelle van Camp, Francoise le Marrec-Croq, Jacopo Vizioli and Pierre-Eric Sauti&#232;re for their strong contribution to the scientific and technical developments.

1. Primary Culture of Microglia/Macrophages
<ol>
	<li>Primary culture of microglia
	<ol>
		<li>Culture commercial rat primary microglia (2 x 106 cells) (see the Table of Materials) in Dulbecco&#39;s modified Eagle medium (DMEM) supplemented with 10% exosome-free serum, 100 U/mL of penicillin, 100 &#181;g/mL streptomycin, and 9.0 g/L glucose at 37 &#176;C and 5% CO2.</li>
		<li>Collect the conditioned medium after a 48 h culture and proceed to the isolation of EVs.</li>
	</ol>
	</li>
	<li>Primary culture of macrophages
	<ol>
		<li>Culture commercial rat primary macrophages (1 x 106 cells) in the medium provided by the manufacturer (see the Table of Materials) at 37 &#176;C and 5% CO2, with exosome-free serum.</li>
		<li>Collect the conditioned medium after a 24 h culture and proceed to the isolation of EVs.</li>
	</ol>
	</li>
</ol>
2. Isolation of EVs
<ol>
	<li>Pre-isolation of EVs from conditioned medium
	<ol>
		<li>Transfer the conditioned culture medium from microglia or macrophage cultures (steps 1.1.2 or 1.2.2) into a conical tube.</li>
		<li>Centrifuge at 1,200 x g for 10 min at room temperature (RT) to pellet the cells.</li>
		<li>Transfer the supernatant into a new conical tube. Centrifuge at 1,200 x g for 20 min at RT to eliminate apoptotic bodies.</li>
		<li>Transfer the supernatant into a 10.4 mL polycarbonate tube and transfer the tube into a 70.1 Ti rotor. Ultracentrifuge at 100,000 x g for 90 min at 4 &#176;C to pellet the EVs.</li>
		<li>Discard the supernatant and resuspend the pellet containing EVs in 200 &#181;L of 0.20 &#181;m filtered phosphate buffer saline (PBS).</li>
	</ol>
	</li>
	<li>Isolation of EVs
	<ol>
		<li>Preparation of the home-made size exclusion chromatography column (SEC)
		<ol>
			<li>Empty a glass chromatography column (length: 26 cm; diameter: 0.6 cm) (see the Table of Materials), wash and sterilize it.</li>
			<li>Place a 60 &#181;m filter at the bottom of the column.</li>
			<li>Stack the column with cross-linked agarose gel filtration base matrix to create a stationary phase of a 0.6 cm diameter and a 20 cm height.</li>
			<li>Rinse the phase with 50 mL of 0.20 &#181;m filtered PBS. Store at 4 &#176;C to be used later if necessary.</li>
		</ol>
		</li>
		<li>Place the resuspended EV pellet on top of the stationary phase of the SEC column.</li>
		<li>Collect 20 sequential fractions of 250 &#181;L while continuing to add 0.20 &#181;m filtered PBS on the top of stationary phase to prevent drying of the column. Store the fractions at -20 &#176;C if necessary. 
		NOTE: A longer storage than one week can be performed at -80 &#176;C to maintain the EV integrity for molecular analysis.</li>
	</ol>
	</li>
	<li>Matrix assisted laser desorption ionization (MALDI) mass spectrometry analysis of the SEC fractions
	<ol>
		<li>Proceed to the EV isolation as described in section 2.2.</li>
		<li>Resuspend the EV pellet with 200 &#181;L of peptide calibration mix solution (see the Table of Materials).</li>
		<li>Proceed to EV collection as described in sections 2.2.1 to 2.2.3.</li>
		<li>Completely dry the fraction with a vacuum concentrator.</li>
		<li>Reconstitute the fractions with 10 &#181;L of 0.1% trifluoroacetic acid (TFA).</li>
		<li>Mix 1 &#181;L of reconstituted fraction with 1 &#181;L of &#945;-cyano-4-hydroxycinnamic acid (HCCA) matrix on a MALDI polished steel target plate.</li>
		<li>Analyze all fractions with a MALDI mass spectrometer.</li>
		<li>Analyze generated spectra with dedicated software (see Table of Materials).</li>
	</ol>
	</li>
</ol>
3. Characterization of EVs
<ol>
	<li>Nanoparticle tracking analysis (NTA) 
	NOTE: The NTA analysis is performed with a nanoparticle tracking analysis instrument (see the Table of Materials) and an automated syringe pump.
	<ol>
		<li>Make a dilution (range of 1:50 to 1:500) of each SEC fraction from step 2.2.3 with 0.20 &#181;m filtered PBS.</li>
		<li>Vortex the solution to eliminate EV aggregates.</li>
		<li>Put the diluted solution in a 1 mL syringe and place it in the automated syringe pump.</li>
		<li>Adjust Camera setting to screen gain level (3) and camera level (13).</li>
		<li>Click on Run and launch the following script.
		<ol>
			<li>Load the sample in analysis chamber (infusion rate: 1,000 for 15 s).</li>
			<li>Decrease and stabilize speed flow for video recording (infusion rate: 25 for 15 s). Capture three consecutive 60 s videos of the particle flow.</li>
			<li>Adjust the camera level (13) and the detection threshold (3) before videos analysis. Click on Settings| Ok to start the analysis and click on Export when the analysis is done.</li>
		</ol>
		</li>
		<li>Between each fraction analysis, wash with 1 mL of 0.20 &#181;m filtered PBS.</li>
	</ol>
	</li>
	<li>Electron microscopy (EM) analysis
	<ol>
		<li>Isolate EVs as described in section 2. 
		NOTE: Sterile conditions are not required.</li>
		<li>Repeat section 3.1 to quantify EVs. 
		NOTE: Only positive fractions will be used for EM analysis.</li>
		<li>Use a 50 kDa centrifugal filter (see Table of Materials) to concentrate EV-containing SEC fractions.</li>
		<li>Resuspend concentrated EVs in 30 &#181;L of 2% paraformaldehyde (PFA).</li>
		<li>Load 10 &#181;L of the sample onto a carbon-coated copper grid.</li>
		<li>Incubate for 20 min in a wet environment.</li>
		<li>Repeat steps 3.2.5 and 3.2.6 for a good absorption of the sample on the grid.</li>
		<li>Transfer the grid into a drop of 1% glutaraldehyde in PBS for 5 min at RT.</li>
		<li>Wash the sample with ultrapure water several times.</li>
		<li>Contrast the sample for 10 min on ice with a mixture of 4% uranyl acetate and 2% methylcellulose (1:9, v/v). Remove excess of the mixture using a filter paper.</li>
		<li>Dry the sample and observe it under a transmission electron microscope at 200 kV (see the Table of Materials).</li>
	</ol>
	</li>
	<li>Western blot analysis
	<ol>
		<li>Protein extraction
		<ol>
			<li>Repeat the steps 3.2.1 to 3.2.3 to isolate and concentrate EVs.</li>
			<li>Mix 50 &#181;L of RIPA buffer (150 mM sodium chloride [NaCl], 50 mM Tris, 5 mM Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid [EGTA], 2 mM Ethylenediamine-tetraacetic acid [EDTA], 100 mM sodium fluoride [NaF], 10 mM sodium pyrophosphate, 1% Nonidet P-40, 1 mM Phenylmethanesulfonyl fluoride [PMSF], 1x protease inhibitor) with the EV sample (25 &#181;L resulting from the EV concentration on filter) for 5 min on ice to extract proteins.</li>
			<li>Sonicate for 5 s (amplitude: 500 W; frequency: 20 kHz), 3 times on ice.</li>
			<li>Remove vesicular debris by centrifugation at 20,000 x g for 10 min, at 4 &#176;C.</li>
			<li>Collect the supernatant and measure the protein concentration with Bradford protein assay method.</li>
		</ol>
		</li>
		<li>Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting
		<ol>
			<li>Mix protein extracts (30 &#181;g) with 5c Laemmli sample buffer (v/v).</li>
			<li>Load the protein mix on a 12% polyacrylamide gel.</li>
			<li>Migrate the proteins in the gel with TGS buffer (25 mM Tris pH 8.5, 192 mM Glycine, and 0.1% SDS), at 70 V for 15 min and 120 V for 45 min.</li>
			<li>Transfer the proteins onto nitrocellulose membrane with semi-dry system at 230 V for 30 min.</li>
			<li>Saturate the membrane for 1 h at RT with blocking buffer (0.05% Tween 20 w/v, 5% milk powder w/v in 0.1 M PBS, pH 7.4).</li>
			<li>Incubate the membrane overnight at 4 &#176;C with mouse monoclonal anti-human heat-shock protein 90 (HSP90) antibody diluted in blocking buffer (1:100).</li>
			<li>Wash the membrane three times with PBS-Tween (PBS, 0.05% Tween 20 w/v) for 15 min.</li>
			<li>Incubate the membrane for 1 h at RT with goat horseradish peroxidase-conjugated anti-mouse IgG secondary antibody diluted in blocking buffer (1:10,000). 
			NOTE: A negative control is performed using secondary antibody alone.</li>
			<li>Repeat the washing step (step 3.3.2.7).</li>
			<li>Reveal the membrane with enhanced chemiluminescence (ECL) western blotting substrate kit (see Table of Materials).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Proteomic analysis
	<ol>
		<li>Protein extraction and in-gel digestion
		<ol>
			<li>Repeat the steps 3.2.1 to 3.2.3 to isolate and concentrate the EVs. Repeat step 3.3.1 for EV protein extraction.</li>
			<li>Perform protein migration in the stacking gel of a 12% polyacrylamide gel.</li>
			<li>Fix the proteins in the gel with coomassie blue for 20 min at RT.</li>
			<li>Excise each colored gel piece and cut it into small pieces of 1 mm3.</li>
			<li>Wash the gel pieces successively with 300 &#181;L of each solutions: ultrapure water for 15 min, 100% acetonitrile (ACN) for 15 min, 100 mM ammonium bicarbonate (NH4HCO3) for 15 min, ACN:100 mM NH4HCO3 (1:1, v/v) for 15 min, and 100% ACN for 5 min with continuous stirring.</li>
			<li>Dry completely gel pieces with vacuum concentrator.</li>
			<li>Perform protein reduction with 100 &#181;L of 100 mM NH4HCO3 containing 10 mM dithiothreitol for 1 h at 56&#176;C.</li>
			<li>Perform protein alkylation with 100 &#181;L of 100 mM NH4HCO3 containing 50 mM iodoacetamide for 45 min in the dark at RT.</li>
			<li>Wash the gel pieces successively with 300 &#181;L of each solution: 100 mM of NH4HCO3 for 15 min, ACN:20 mM NH4HCO3 (1:1, v/v) for 15 min, and 100% ACN for 5 min with a continuous stirring.</li>
			<li>Completely dry the gel pieces with a vacuum concentrator.</li>
			<li>Perform protein digestion with 50 &#181;L of trypsin (12.5 &#181;g/mL) in 20 mM NH4HCO3 overnight at 37 &#176;C.</li>
			<li>Extract the digested proteins from the gel with 50 &#181;L of 100% ACN for 30 min at 37 &#176;C and then 15 min at RT with continuous stirring.</li>
			<li>Repeat the following extraction procedures twice: 50 &#181;L of 5% TFA in 20 mM NH4HCO3 solution for 20 min with continuous stirring.</li>
			<li>Add 100 &#181;L of 100% ACN for 10 min with continuous stirring.</li>
			<li>Dry digested proteins with a vacuum concentrator and resuspend in 20 &#181;L of 0.1% TFA.</li>
			<li>Desalt the sample using a 10 &#181;L pipette tip with C18 reverse phase media for desalting and concentrating peptides (see the Table of Materials) and elute peptides with ACN:0.1% formic acid (FA) (80:20, v/v).</li>
			<li>Completely dry the sample with a vacuum concentrator and resuspend in 20 &#181;L of ACN:0.1% FA (2:98, v/v) for liquid chromatography tandem mass spectrometry (LC-MS/MS).</li>
		</ol>
		</li>
		<li>LC-MS/MS Analysis
		<ol>
			<li>Load the digested peptide into the LC-MS/MS instrument and perform sample and data analysis according to parameters described in detail elsewhere42.</li>
		</ol>
		</li>
		<li>Raw data analysis
		<ol>
			<li>Process mass spectrometry data to identify proteins and compare identified proteins of each sample with a quantitative proteomics software package using standard parameters.</li>
			<li>Export, using standard parameters, the list of exclusive and over-represented proteins from EV positive samples in a software predicting protein networks and biological processes.</li>
			<li>Compare the list of identified proteins in the fractions with the top 100 EV markers from the Exocarta open access database (see the Table of Materials).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Functional EVs Effects Assay
<ol>
	<li>Neurite outgrowth assay on PC-12 cell line
	<ol>
		<li>Culture PC12 cell line in complete DMEM medium (2 mM L-glutamine, 10% fetal horse serum (FHS), 5% fetal bovine serum (FBS), 100 UI/mL penicillin, 100 &#181;g/mL streptomycin). 
		NOTE: The sera are exosome free in the whole procedure.</li>
		<li>Add a cover glass (all wells) to a 24-well plate; coat the plate with poly-D-lysine (0.1 mg/mL) and seed 260,000 cells/well.</li>
		<li>Incubate the cells at 37 &#176;C under 5% CO2.</li>
		<li>After 24 h of incubation, change the medium to DMEM differentiation medium (DMEM with 2 mM L-glutamine, 0.1% FHS, 100 UI/mL penicillin, 100 &#181;g/mL streptomycin) with 1 x 106 microglia EVs (from step 1.1.2). 
		NOTE: Control condition is performed without microglia EVs in the differentiation medium.</li>
		<li>At day 4 after seeding, load all wells with 100 &#181;L of complete DMEM medium.</li>
		<li>At day 7 after seeding, fix the cells with 4% PFA for 20 min at RT and rinse three times (10 min each) with PBS.</li>
		<li>Stain the cells with rhodamine-conjugated phalloidin for 30 min at 4 &#176;C and rinse 3 times (10 min each) with PBS.</li>
		<li>Stain the cells with diluted Hoechst 33342 (1:10,000) for 30 min at RT and rinse 3 times (10 min each) with PBS.</li>
		<li>Mount the cover glass on a slide with fluorescent mounting medium (see Table of Materials).</li>
		<li>Analyze the slide with a confocal microscope, take 5 random images of each slide.</li>
		<li>Measure neurite outgrowth using an automated quantification software (total neurite length) as described in detail elsewhere43.</li>
	</ol>
	</li>
	<li>Neurite outgrowth on rat primary neurons
	<ol>
		<li>Coat an 8-well glass slide with poly-D-lysine (0.1 mg/mL) and laminin (20 &#181;g/mL).</li>
		<li>Culture rat commercial primary neurons in appropriate culture medium (see Table of Materials) by plating 50,000 cells per well and incubating the cells at 37 &#176;C under 5% CO2 for 48 h. 
		NOTE: The sera are exosome-free in the whole procedure.</li>
		<li>Add 1 x 106 microglia EVs to neuron culture medium and incubate at 37 &#176;C under 5% CO2 for 48 h more. 
		NOTE: Control condition is performed without microglia EVs in neuron culture medium.</li>
		<li>Follow steps 4.1.6 to 4.1.9 to fix and stain the cells.</li>
		<li>Follow steps 4.1.10 and 4.1.11 to analyze the slide.</li>
	</ol>
	</li>
	<li>Glioma cell invasion
	<ol>
		<li>Resuspend C6 rat glioma cells in complete DMEM medium (DMEM with 10% FBS, 2 mM L-glutamine, 1x antibiotics) containing 5% collagen at a final concentration of 8,000 cells in 20 &#181;L. 
		NOTE: The sera are exosome-free in the whole procedure.</li>
		<li>Place 5 mL of PBS AT the bottom of a 60 mm tissue culture dish. Invert the lid and deposit drops of 20 &#181;L (8,000 cells) of cell suspension onto the bottom of the lid.</li>
		<li>Invert the lid onto the PBS-filled bottom chamber and incubate the plate at 37 &#176;C and 5% CO2 for 72 h until cell spheroids are formed.</li>
		<li>Add 1 x 108 macrophage EVs (from step 1.2.2) to a 2.2 mg/mL collagen mixture (2 mL of bovine collagen type I solution [3 mg/mL] with 250 &#181;L of 10x minimum essential medium (MEM) and 500 &#181;L of 0.1 M sodium hydroxide). 
		NOTE: Control condition is performed without macrophage EVs in the collagen mixture.</li>
		<li>Distribute the collagen mixture containing EVs in a 24-well plate for embedding cell spheroids.</li>
		<li>Implant the newly formed cell spheroids at the center of each well.</li>
		<li>Incubate the plate for 30 min at 37&#176;C and 5% CO2 to solidify the gel.</li>
		<li>Thereafter, overlay 400 &#181;L of complete DMEM medium on the collagen matrix in each well.</li>
		<li>Incubate the complete system for a total of 6 days at 37 &#176;C and 5% CO2. 
		NOTE: Cell invasion out of the spheroid is monitored by digital photography using an inverted light microscope using a 4x/0.10 N.A. objective.</li>
		<li>Acquire images of each well every day.</li>
		<li>Process images and quantify invasion of cell spheroid areas using the software as previously described in detail42. 
		NOTE: Invasion and spheroid areas were normalized for each day to the invasion and spheroid areas measured on day 0.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

The central nervous system (CNS) is a complex tissue in which cell-to-cell communication regulates normal neuronal functions necessary for homeostasis30. EVs are now widely studied and described as important molecular cargos for cell-to-cell communication31. They specifically deliver a cocktail of mediators to recipient cells thereby affecting their functions in healthy and pathological conditions32. Recent studies indicate that EVs play a crucial ro...

Many neuropathologies are related to the neuro-inflammatory state which is a complex mechanism that is increasingly considered, but still poorly understood because the immune processes are diverse and depend upon the cell environment. Indeed, the CNS disorders do not systematically involve the same activation signals and immune cell populations and thus the pro- or anti-inflammatory responses are difficult to evaluate as causes or consequences of pathologies. The brain resident macrophages called &#8220;microglia&#8221; appear to be at the interface between the nervous and immune systems1. Microglia have a myeloid origin and are derived from the yolk sac during primitive hematopoiesis to colonize the brain, whereas peripheral macrophages are derived from the fetal liver during definitive hematopoiesis to become peripheral macrophages2. The microglia cells communicate with neurons and neuron-derived glial cells such as astrocytes and oligodendrocytes3. Several recent studies have demonstrated that microglia are involved in neuronal plasticity during CNS development and adult tissue homeostasis, and also in the inflammatory state associated with neurodegenerative diseases4,5. Otherwise, the integrity of the blood brain barrier can be compromised in other CNS pathologies. The immune responses, especially in the glioblastoma multiforme cancer, are not supported only by microglia cells as the blood brain barrier is reorganized through angiogenic processes and the presence of lymphatic vessels6,7. Therefore, a large bone marrow-derived macrophages (BMDMs) infiltration occurs in the brain tumor throughout tumor-dependent angiogenesis mechanisms8. The cancer cells exert a significant influence on infiltrated BMDMs leading to immunosuppressive properties and tumor growth9. Thus the communication between the immune cells and their brain microenvironment is difficult to understand as the cell origin and activation signals are diverse10,11. It is thus interesting to apprehend the functions of immune cell-associated molecular signatures in physiological conditions. In this regard, the cell-cell communication between immune cells and cell microenvironment can be studied through the release of extracellular vesicles (EVs).
The EVs are being studied more and more in the regulation of immune functions in healthy as well as pathological conditions12,13. Two populations, exosomes and microvesicles, can be taken into account. They present different biogenesis and size ranges. The exosomes are vesicles of ~30&#8211;150 nm diameter and are generated from the endosomal system and secreted during fusion of multivesicular bodies (MVBs) with the plasma membrane. The microvesicles are about 100&#8211;1,000 nm in diameter and are generated by an outward budding from the cell plasma membrane14. Because the exosome versus microvesicle discrimination is still difficult to realize according to the size and molecular patterns, we will only use the term EVs in the present report. The EV-associated communication in the CNS represents an ancestral mechanism since studies showed their involvement in invertebrate species including nematodes, insects or annelids15,16. Moreover, the results showing that EVs can communicate with cells from different species demonstrate this mechanism to be a key-lock system, based first on surface-molecule recognition between vesicles and recipient cells and then allowing the uptake of mediators16,17. Indeed, the EVs contain many molecules like proteins (e.g., enzymes, signal transduction, biogenesis factor), lipids (e.g., ceramide, cholesterol) or nucleic acids (e.g., DNA, mRNA or miRNAs) acting as direct or indirect regulators of the recipient cell activities14. That is why methodological studies were also performed on immune cells to isolate EVs and fully characterize their protein signatures18,19.
The earliest studies demonstrated the release of exosomes from primary cultured rat microglia as an inducible mechanism following a Wnt3a- or serotonin-dependent activation20,21. Functionally in the CNS, microglia-derived EVs regulate the synaptic vesicle release by presynaptic terminals in neurons contributing to the control of the neuronal excitability22,23. Microglia-derived EVs could also propagate cytokines-mediated inflammatory response in large brain regions24,25. Importantly, the diverse ligands for toll-like receptor family might activate specific productions of EVs in the microglia26. For example, in vitro studies show that LPS-activated microglia BV2 cell lines produce differential EV contents including pro-inflammatory cytokines27. Therefore, the functional diversity of immune cell subpopulations in the CNS, microglia and infiltrating BMDMs, might be evaluated through their own EV populations including the EV impact on recipient cells and the identification of EV contents.
We previously described methods to evaluate the functional properties of microglia- and BMDM-derived EVs after their isolation16,19. In the present report, we propose to independently evaluate the effect of microglia-derived EVs on neurite outgrowth, and the effect of macrophage-derived EVs on the control of glioma cell aggregates. This study also proposes a wide proteomic analysis of the EV fractions in order to validate the EV isolation procedure as well as identify the biologically active protein signatures. The beneficial effects and the molecular deciphering of EV contents could help their possible manipulation and use as therapeutic agents in brain disorders.

<table><tbody><tr><td>12% Mini-PROTEAN&amp;nbsp;TGX Precast Protein Gels</td><td>Bio-rad</td><td>4561045EDU</td><td>&amp;nbsp;</td></tr><tr><td>Acetonitrile</td><td>Fisher Chemicals</td><td>A955-1</td><td>&amp;nbsp;</td></tr><tr><td>Amicon 50 kDa centrifugal filter</td><td>Merck</td><td>UFC505024</td><td>&amp;nbsp;</td></tr><tr><td>Ammonium bicarbonate</td><td>Sigma-Aldrich</td><td>9830</td><td>&amp;nbsp;</td></tr><tr><td>HSP90 &amp;alpha;/&amp;beta; antibody (RRID: AB_675659)</td><td>Santa-cruz</td><td>sc-13119</td><td>&amp;nbsp;</td></tr><tr><td>B27 Plus supplement</td><td>Gibco</td><td>A3582801</td><td>&amp;nbsp;</td></tr><tr><td>BenchMixer V2 Vortex Mixer</td><td>Benchmark Scientific</td><td>BV1003</td><td>&amp;nbsp;</td></tr><tr><td>Bio-Rad Protein Assay Dye Reagent Concentrate (Bradford)</td><td>Bio-Rad</td><td>5000006</td><td>&amp;nbsp;</td></tr><tr><td>C18 ZipTips</td><td>Merck Millipore</td><td>ZTC18S096</td><td>&amp;nbsp;</td></tr><tr><td>C6 rat glioma cell</td><td>ATCC</td><td>ATCC CCL-107</td><td>&amp;nbsp;</td></tr><tr><td>Canonical tubes</td><td>Sarstedt</td><td>62.554.002</td><td>&amp;nbsp;</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804000010</td><td>&amp;nbsp;</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>ThermoFisher</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Confocal microscope LSM880</td><td>Carl Zeiss</td><td>LSM880</td><td>&amp;nbsp;</td></tr><tr><td>Cover glass</td><td>Marienfeld</td><td>111580</td><td>&amp;nbsp;</td></tr><tr><td>Culture Dish (60 mm)</td><td>Sarstedt</td><td>82.1473</td><td>&amp;nbsp;</td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>43819</td><td>&amp;nbsp;</td></tr><tr><td>DMEM</td><td>Gibco</td><td>41966029</td><td>&amp;nbsp;</td></tr><tr><td>EASY-nLC 1000 Liquid Chromatograph</td><td>ThermoFisher</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Electron microscope JEM-2100</td><td>JEOL</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&amp;prime;,N&amp;prime;-tetraacetic acid</td><td>Sigma-Aldrich</td><td>03777-10G</td><td>&amp;nbsp;</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma-Aldrich</td><td>ED-100G</td><td>&amp;nbsp;</td></tr><tr><td>Exo-FBS</td><td>Ozyme</td><td>EXO-FBS-50A-1</td><td>Exosome depleted FBS</td></tr><tr><td>ExoCarta database (top 100 proteins of Evs)</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>http://www.exocarta.org/</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>16140071</td><td>&amp;nbsp;</td></tr><tr><td>Fetal Horse Serum</td><td>Biowest</td><td>S0960-500</td><td>&amp;nbsp;</td></tr><tr><td>Filtropur S 0.2</td><td>Sarstedt</td><td>83.1826.001</td><td>&amp;nbsp;</td></tr><tr><td>Fisherbrand&amp;nbsp;Q500 Sonicator with Probe</td><td>Fisherbrand</td><td>12893543</td><td>&amp;nbsp;</td></tr><tr><td>FlexAnalysis</td><td>Brucker</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Fluorescence mounting medium</td><td>Agilent</td><td>S3023</td><td>&amp;nbsp;</td></tr><tr><td>Formic Acid</td><td>Sigma-Aldrich</td><td>695076</td><td>&amp;nbsp;</td></tr><tr><td>Formvar-carbon coated copper grids</td><td>Agar scientific Ltd</td><td>AGS162-3</td><td>&amp;nbsp;</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8769</td><td>&amp;nbsp;</td></tr><tr><td>Glutaraldehyde</td><td>Sigma-Aldrich</td><td>340855</td><td>&amp;nbsp;</td></tr><tr><td>Hoechst 33342</td><td>Euromedex</td><td>17535-AAT</td><td>&amp;nbsp;</td></tr><tr><td>Idoacetamide</td><td>Sigma-Aldrich</td><td>I1149</td><td>&amp;nbsp;</td></tr><tr><td>InstantBlue Coomassie Protein Stain</td><td>Expedeon</td><td>ISB1L</td><td>&amp;nbsp;</td></tr><tr><td>Invert light microscope CKX53</td><td>Olympus</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030-024</td><td>&amp;nbsp;</td></tr><tr><td>LabTek II 8 wells</td><td>&amp;nbsp;Nunc</td><td>154534</td><td>&amp;nbsp;</td></tr><tr><td>Laemmli 2x</td><td>Bio-Rad</td><td>1610737</td><td>&amp;nbsp;</td></tr><tr><td>Laminin</td><td>Corning</td><td>354232</td><td>&amp;nbsp;</td></tr><tr><td>MaxQuant software (proteins identification software)</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>https://maxquant.net/maxquant/</td></tr><tr><td>MBT Polish stell</td><td>Brucker</td><td>8268711</td><td>&amp;nbsp;</td></tr><tr><td>MEM 10x</td><td>Gibco</td><td>21090-022</td><td>&amp;nbsp;</td></tr><tr><td>Methylcellulose</td><td>Sigma-Aldrich</td><td>M6385-100G</td><td>&amp;nbsp;</td></tr><tr><td>MiliQ water</td><td>Merck Millipore</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Milk</td><td>Regilait</td><td>REGILAIT300</td><td>&amp;nbsp;</td></tr><tr><td>Mini PROTEAN Vertical Electrophoresis Cell</td><td>Bio-Rad</td><td>1658000FC</td><td>&amp;nbsp;</td></tr><tr><td>MonoP FPLC column</td><td>GE Healthcare</td><td>&amp;nbsp;</td><td>no longer available</td></tr><tr><td>Nanosight NS300</td><td>Malvern Panalytical</td><td>NS300</td><td>&amp;nbsp;</td></tr><tr><td>NanoSight NTA software v3.2</td><td>Malvern Panalytical</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>NanoSight syringe pump</td><td>Malvern Panalytical</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Neurobasal</td><td>Gibco</td><td>21103-049</td><td>&amp;nbsp;</td></tr><tr><td>Nitrocellulose membrane</td><td>GE Healthcare</td><td>10600007</td><td>&amp;nbsp;</td></tr><tr><td>Nonidet P-40</td><td>Fluka</td><td>56741</td><td>&amp;nbsp;</td></tr><tr><td>Nunc multidish 24 wells</td><td>ThermoFisher</td><td>82.1473</td><td>&amp;nbsp;</td></tr><tr><td>Paraformaldehyde</td><td>Electro microscopy Science</td><td>15713</td><td>&amp;nbsp;</td></tr><tr><td>PC-12 cell line</td><td>ATCC</td><td>ATCC CRL-1721</td><td>&amp;nbsp;</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td>&amp;nbsp;</td></tr><tr><td>Peptide calibration mix</td><td>LaserBio Labs</td><td>C101</td><td>&amp;nbsp;</td></tr><tr><td>Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>115-035-003</td><td>&amp;nbsp;</td></tr><tr><td>Perseus software (Processing of identified proteins)</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>https://maxquant.net/perseus/</td></tr><tr><td>Phalloidin-tetramethylrhodamine conjugate</td><td>Santa-cruz</td><td>sc-362065</td><td>&amp;nbsp;</td></tr><tr><td>Phenylmethanesulfonyl fluoride</td><td>Sigma-Aldrich</td><td>78830</td><td>&amp;nbsp;</td></tr><tr><td>Phosphate Buffer Saline</td><td>Invitrogen</td><td>14190094</td><td>no calcium, no magnesium</td></tr><tr><td>pluriStrainer M/ 60 &amp;micro;m</td><td>pluriSelect</td><td>43-50060</td><td>&amp;nbsp;</td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich</td><td>P6407</td><td>&amp;nbsp;</td></tr><tr><td>Polycarbonate centrifuge tubes</td><td>Beckman Coulter</td><td>355651</td><td>&amp;nbsp;</td></tr><tr><td>Protease Inhibitor</td><td>Sigma-Aldrich</td><td>S8830-20TAB</td><td>&amp;nbsp;</td></tr><tr><td>PureCol</td><td>Cell Systems</td><td>5005</td><td>&amp;nbsp;</td></tr><tr><td>Q-Exactive mass spectrometer</td><td>ThermoFisher</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>rapifleX mass spectrometer</td><td>Brucker</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Rat cortical neurons</td><td>Cell Applications</td><td>R882N-20</td><td>Cell origin : Derived from cerebral cortices of day 18 embryonic Sprague Dawley rat brains</td></tr><tr><td>Rat Macrophage &amp;amp; Microglia Culture Medium</td><td>Cell Applications</td><td>R620K-100</td><td>Cell orgin : Normal healthy Rat bone marrow</td></tr><tr><td>Rat primary macrophages</td><td>Cell Applications</td><td>R8818-10a</td><td>&amp;nbsp;</td></tr><tr><td>Rat primary microglia</td><td>Lonza</td><td>RG535</td><td>&amp;nbsp;</td></tr><tr><td>Sepharose CL-2B</td><td>GE Healthcare</td><td>17014001</td><td>&amp;nbsp;</td></tr><tr><td>Sequencing Grade Modified Trypsin</td><td>Promega</td><td>V5111</td><td>&amp;nbsp;</td></tr><tr><td>Slide</td><td>Dustsher</td><td>100204</td><td>&amp;nbsp;</td></tr><tr><td>Sodium Chloride</td><td>Scharlau</td><td>SO0227</td><td>&amp;nbsp;</td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>Sigma-Aldrich</td><td>L3771</td><td>&amp;nbsp;</td></tr><tr><td>Sodium Fluoride</td><td>Sigma-Aldrich</td><td>S7920-100G</td><td>&amp;nbsp;</td></tr><tr><td>Sodium hydroxide</td><td>Scharlab</td><td>SO0420005P</td><td>&amp;nbsp;</td></tr><tr><td>Sodium pyrophosphate</td><td>Sigma-Aldrich</td><td>S6422-100G</td><td>&amp;nbsp;</td></tr><tr><td>SpeedVac Vacuum Concentrator</td><td>ThermoFisher</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>String software (functional protein association networks)</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>https://string-db.org/</td></tr><tr><td>SuperSignal West Dura extended Duration Substrate</td><td>ThermoFisher</td><td>34075</td><td>&amp;nbsp;</td></tr><tr><td>Syringe 1.0 mL</td><td>Terumo</td><td>8SS01H1</td><td>&amp;nbsp;</td></tr><tr><td>Trans-Blot SD Semi-Dry Transfer cell</td><td>Bio-Rad</td><td>1703940</td><td>&amp;nbsp;</td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma-Aldrich</td><td>T6508</td><td>&amp;nbsp;</td></tr><tr><td>Tris</td><td>Interchim</td><td>UP031657</td><td>&amp;nbsp;</td></tr><tr><td>Tris-Glycine</td><td>Euromedex</td><td>EU0550</td><td>&amp;nbsp;</td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P2287</td><td>&amp;nbsp;</td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td>A95765</td><td>&amp;nbsp;</td></tr><tr><td>Ultracentrifuge Rotor 70.1 Ti</td><td>Beckman Coulter</td><td>342184</td><td>&amp;nbsp;</td></tr><tr><td>Uranyl acetate</td><td>Agar Scientific Ltd</td><td>AGR1260A</td><td>&amp;nbsp;</td></tr><tr><td>Whatman filter paper</td><td>Sigma-Aldrich</td><td>WHA10347510</td><td>&amp;nbsp;</td></tr><tr><td>&amp;alpha;-Cyano-4-hydroxycinnamic acid</td><td>Sigma-Aldrich</td><td>C2020-25G</td><td>&amp;nbsp;</td></tr></tbody></table>

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.

One of the main challenges to attributing biologicals effects to extracellular vesicles (EVs) is the ability to isolate the EVs from the whole culture medium. In this report, we present a method using ultracentrifugation (UC) and size-exclusion chromatography (SEC) which is coupled to the large-scale analysis of protein signatures to validate EV markers and identify bioactive compounds. The macrophage- or microglia-derived EVs were isolated from the conditioned medium after a 24 h or 48 h culture respectively (<strong cl...

Watch this Scientific Journal Video about Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment at JoVE.com

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

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 ...

characterization-immune-cell-derived-extracellular-vesicles-studying

Research

JoVE Journal

Neuroscience

6.8K Views.  Univ. Lille, INSERM. 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.

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

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

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

Biomanufacturing and Characterization of NK-Extracellular Vesicles

Scalable Biomanufacturing Workflow to Produce and Isolate Natural Killer Cell-Derived Extracellular Vesicle-Based Cancer Biotherapeutics

Natural killer cell-derived extracellular vesicles (NK-EVs) are being investigated as cancer biotherapeutics. They possess unique properties as cytotoxic nanovesicles targeting cancer cells and as immunomodulatory communicators. A scalable biomanufacturing workflow enables the production of large quantities of high-purity NK-EVs to meet the pre-clinical and clinical demands. The workflow employs a closed-loop hollow-fiber bioreactor, enabling continuous production of NK-EVs from the NK92-MI cell line under serum-free, xeno-free, feeder-free, and antibiotic-free conditions in compliance with Good Manufacturing Practices standards. This protocol-driven study outlines the biomanufacturing workflow for isolating NK-EVs using size-exclusion chromatography, ultrafiltration, and filter-based sterilization. Essential NK-EV product characterization is performed via nanoparticle tracking analysis, and their functionality is assessed through a validated cell viability-based potency assay against cancer cells. This scalable biomanufacturing process holds significant potential to advance the clinical translation of NK-EV-based cancer biotherapeutics by adhering to best practices and ensuring reproducibility.

Natural killer cell-derived extracellular vesicles (NK-EVs) hold promising potential as cancer biotherapeutics. This methodology-based study presents a scalable closed-loop biomanufacturing workflow designed to continuously produce and isolate large quantities of high-purity NK-EVs. In-process control testing is performed throughout the biomanufacturing workflow, ensuring the EVs meet quality standards for product release.

Natural killer cell-derived extracellular vesicles (NK-EVs) are being investigated as cancer biotherapeutics. They possess unique properties as cytotoxic ...

Evaluation of the Storage Stability of Extracellular Vesicles

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical development, not only their pharmaceutical activity is important but also their production needs to be evaluated. In this context, research focuses on the isolation of EVs, their characterization, and their storage. The present manuscript aims at providing a facile procedure for the assessment of the effect of different storage conditions on EVs, without genetic manipulation or specific functional assays. This makes it possible to quickly get a first impression of the stability of EVs under a given storage condition, and EVs derived from different cell sources can be compared easily. The stability measurement is based on the physicochemical parameters of the EVs (size, particle concentration, and morphology) and the preservation of the activity of their cargo. The latter is assessed by the saponin-mediated encapsulation of the enzyme beta-glucuronidase into the EVs. Glucuronidase acts as a surrogate and allows for an easy quantification via the cleavage of a fluorescent reporter molecule. The present protocol could be a tool for researchers in the search for storage conditions that optimally retain EV properties to advance EV research toward clinical application.

Here we present a readily applicable protocol to assess the storage stability of extracellular vesicles, a group of naturally occurring nanoparticles produced by cells. The vesicles are loaded with glucuronidase as a model enzyme and stored under different conditions. After storage, their physicochemical parameters and the activity of the encapsulated enzyme are evaluated.

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical ...

Bioengineering

Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and significant role as carriers of biological information make them intriguing study objects, requiring reliable and repetitive protocols for their isolation. However, realizing their full potential is difficult as there are still many technical obstacles related to their research (like proper acquisition). This study presents a protocol for the isolation of small EVs (according to the MISEV 2018 nomenclature) from the culture supernatant of tumor cell lines based on differential centrifugation. The protocol includes guidelines on how to avoid contamination with endotoxins during the isolation of EVs and how to properly evaluate them. Endotoxin contamination of EVs can significantly hinder subsequent experiments or even mask their true biological effects. On the other hand, the overlooked presence of endotoxins may lead to incorrect conclusions. This is of particular importance when referring to cells of the immune system, including monocytes, because monocytes constitute a population that is especially sensitive to endotoxin residues. Therefore, it is highly recommended to screen EVs for endotoxin contamination, especially when working with endotoxin-sensitive cells such as monocytes, macrophages, myeloid-derived suppressor cells, or dendritic cells.

The proposed protocol includes guidelines on how to avoid contamination with endotoxin during the isolation of extracellular vesicles from cell culture supernatants, and how to properly evaluate them.

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and ...

Immunology and Infection

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte-derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances, including obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing,&#160;microarrays and can be&#160;utilized in functional in vitro/in vivo studies.

We describe the isolation of human adipocyte-derived extracellular vesicles (EVs) from gluteal and abdominal adipose tissue using filtration and ultracentrifugation. We characterize the isolated adipocyte-derived EVs by determining their size and concentration by Nanoparticle Tracking Analysis and by western blotting for the presence of EV-protein tumor susceptibility gene 101 (TSG101).

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can ...

Biology

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often require indirect methods due to their small diameter (40-250 nm), which is beneath the diffraction limit of typical light microscopy. We have developed a super-resolution microscopy-based visualization of EVs to bypass the diffraction limit in both two and three dimensions. Using this approach, we can resolve the three-dimensional shape of EVs to within +/- 20 nm resolution on the XY-axis and +/- 50 nm resolution along the Z-axis. In conclusion, we propose that super-resolution microscopy be considered as a characterization method of EVs, including exosomes, as well as enveloped viruses.

Direct stochastic optical reconstruction microscopy (dSTORM) is used to bypass the typical diffraction limit of light microscopy and to view exosomes at the nanometer scale. It can be employed in both two and three dimensions to characterize exosomes.

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often ...

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 derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

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

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

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

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.

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 ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

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

Summary

Explore More Videos

Chapters in this video

Extraction of Extracellular Vesicles from Whole Tissue

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Scalable Biomanufacturing Workflow to Produce and Isolate Natural Killer Cell-Derived Extracellular Vesicle-Based Cancer Biotherapeutics

Evaluation of the Storage Stability of Extracellular Vesicles

Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

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

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

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells