Name: characterization of immune cell-derived extracellular vesicles and studying functional impact on cell environment
Uploaded: 2020-06-02T12:30:00
Description: Watch this Scientific Journal Video about Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment at JoVE.com

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....

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&nbsp;TGX Precast Protein Gels</td>
<td>Bio-rad</td>
<td>4561045EDU</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Acetonitrile</td>
<td>Fisher Chemicals</td>
<td>A955-1</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Amicon 50 kDa centrifugal filter</td>
<td>Merck</td>
<td>UFC505024</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ammonium bicarbonate</td>
<td>Sigma-Aldrich</td>
<td>9830</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>HSP90 &alpha;/&beta; antibody (RRID: AB_675659)</td>
<td>Santa-cruz</td>
<td>sc-13119</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>B27 Plus supplement</td>
<td>Gibco</td>
<td>A3582801</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>BenchMixer V2 Vortex Mixer</td>
<td>Benchmark Scientific</td>
<td>BV1003</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Bio-Rad Protein Assay Dye Reagent Concentrate (Bradford)</td>
<td>Bio-Rad</td>
<td>5000006</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>C18 ZipTips</td>
<td>Merck Millipore</td>
<td>ZTC18S096</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>C6 rat glioma cell</td>
<td>ATCC</td>
<td>ATCC CCL-107</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Canonical tubes</td>
<td>Sarstedt</td>
<td>62.554.002</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Centrifuge</td>
<td>Eppendorf</td>
<td>5804000010</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>CO2 Incubator</td>
<td>ThermoFisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Confocal microscope LSM880</td>
<td>Carl Zeiss</td>
<td>LSM880</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Cover glass</td>
<td>Marienfeld</td>
<td>111580</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Culture Dish (60 mm)</td>
<td>Sarstedt</td>
<td>82.1473</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Dithiothreitol</td>
<td>Sigma-Aldrich</td>
<td>43819</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>DMEM</td>
<td>Gibco</td>
<td>41966029</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>EASY-nLC 1000 Liquid Chromatograph</td>
<td>ThermoFisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Electron microscope JEM-2100</td>
<td>JEOL</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&prime;,N&prime;-tetraacetic acid</td>
<td>Sigma-Aldrich</td>
<td>03777-10G</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ethylenediaminetetraacetic acid</td>
<td>Sigma-Aldrich</td>
<td>ED-100G</td>
<td>&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>&nbsp;</td>
<td>&nbsp;</td>
<td>http://www.exocarta.org/</td>
</tr>
<tr>
<td>Fetal Bovine Serum</td>
<td>Gibco</td>
<td>16140071</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Fetal Horse Serum</td>
<td>Biowest</td>
<td>S0960-500</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Filtropur S 0.2</td>
<td>Sarstedt</td>
<td>83.1826.001</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Fisherbrand&nbsp;Q500 Sonicator with Probe</td>
<td>Fisherbrand</td>
<td>12893543</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>FlexAnalysis</td>
<td>Brucker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Fluorescence mounting medium</td>
<td>Agilent</td>
<td>S3023</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Formic Acid</td>
<td>Sigma-Aldrich</td>
<td>695076</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Formvar-carbon coated copper grids</td>
<td>Agar scientific Ltd</td>
<td>AGS162-3</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Glucose</td>
<td>Sigma-Aldrich</td>
<td>G8769</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Glutaraldehyde</td>
<td>Sigma-Aldrich</td>
<td>340855</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Hoechst 33342</td>
<td>Euromedex</td>
<td>17535-AAT</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Idoacetamide</td>
<td>Sigma-Aldrich</td>
<td>I1149</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>InstantBlue Coomassie Protein Stain</td>
<td>Expedeon</td>
<td>ISB1L</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Invert light microscope CKX53</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>L-glutamine</td>
<td>Gibco</td>
<td>25030-024</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>LabTek II 8 wells</td>
<td>&nbsp;Nunc</td>
<td>154534</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Laemmli 2X</td>
<td>Bio-Rad</td>
<td>1610737</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Laminin</td>
<td>Corning</td>
<td>354232</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>MaxQuant software (proteins identification software)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>https://maxquant.net/maxquant/</td>
</tr>
<tr>
<td>MBT Polish stell</td>
<td>Brucker</td>
<td>8268711</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>MEM 10X</td>
<td>Gibco</td>
<td>21090-022</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Methylcellulose</td>
<td>Sigma-Aldrich</td>
<td>M6385-100G</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>MiliQ water</td>
<td>Merck Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Milk</td>
<td>Regilait</td>
<td>REGILAIT300</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Mini PROTEAN Vertical Electrophoresis Cell</td>
<td>Bio-Rad</td>
<td>1658000FC</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>MonoP FPLC column</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>no longer available</td>
</tr>
<tr>
<td>Nanosight NS300</td>
<td>Malvern Panalytical</td>
<td>NS300</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>NanoSight NTA software v3.2</td>
<td>Malvern Panalytical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>NanoSight syringe pump</td>
<td>Malvern Panalytical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Neurobasal</td>
<td>Gibco</td>
<td>21103-049</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Nitrocellulose membrane</td>
<td>GE Healthcare</td>
<td>10600007</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Nonidet P-40</td>
<td>Fluka</td>
<td>56741</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Nunc multidish 24 wells</td>
<td>ThermoFisher</td>
<td>82.1473</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Paraformaldehyde</td>
<td>Electro microscopy Science</td>
<td>15713</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>PC-12 cell line</td>
<td>ATCC</td>
<td>ATCC CRL-1721</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Penicillin-Streptomycin</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Peptide calibration mix</td>
<td>LaserBio Labs</td>
<td>C101</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L)</td>
<td>Jackson ImmunoResearch</td>
<td>115-035-003</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Perseus software (Processing of identified proteins)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>https://maxquant.net/perseus/</td>
</tr>
<tr>
<td>Phalloidin-tetramethylrhodamine conjugate</td>
<td>Santa-cruz</td>
<td>sc-362065</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Phenylmethanesulfonyl fluoride</td>
<td>Sigma-Aldrich</td>
<td>78830</td>
<td>&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 &micro;m</td>
<td>pluriSelect</td>
<td>43-50060</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Poly-D-lysine</td>
<td>Sigma-Aldrich</td>
<td>P6407</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Polycarbonate centrifuge tubes</td>
<td>Beckman Coulter</td>
<td>355651</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Protease Inhibitor</td>
<td>Sigma-Aldrich</td>
<td>S8830-20TAB</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>PureCol</td>
<td>Cell Systems</td>
<td>5005</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Q-Exactive mass spectrometer</td>
<td>ThermoFisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>rapifleX mass spectrometer</td>
<td>Brucker</td>
<td>&nbsp;</td>
<td>&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; 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>&nbsp;</td>
</tr>
<tr>
<td>Rat primary microglia</td>
<td>Lonza</td>
<td>RG535</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sepharose CL-2B</td>
<td>GE Healthcare</td>
<td>17014001</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sequencing Grade Modified Trypsin</td>
<td>Promega</td>
<td>V5111</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Slide</td>
<td>Dustsher</td>
<td>100204</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sodium Chloride</td>
<td>Scharlau</td>
<td>SO0227</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sodium Dodecyl Sulfate</td>
<td>Sigma-Aldrich</td>
<td>L3771</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sodium Fluoride</td>
<td>Sigma-Aldrich</td>
<td>S7920-100G</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sodium hydroxide</td>
<td>Scharlab</td>
<td>SO0420005P</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sodium pyrophosphate</td>
<td>Sigma-Aldrich</td>
<td>S6422-100G</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>SpeedVac Vacuum Concentrator</td>
<td>ThermoFisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>String software (functional protein association networks)</td>
<td>&nbsp;</td>
<td>&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>&nbsp;</td>
</tr>
<tr>
<td>Syringe 1.0 mL</td>
<td>Terumo</td>
<td>8SS01H1</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Trans-Blot SD Semi-Dry Transfer cell</td>
<td>Bio-Rad</td>
<td>1703940</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Trifluoroacetic acid</td>
<td>Sigma-Aldrich</td>
<td>T6508</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Tris</td>
<td>Interchim</td>
<td>UP031657</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Tris-Glycine</td>
<td>Euromedex</td>
<td>EU0550</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Tween 20</td>
<td>Sigma-Aldrich</td>
<td>P2287</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ultracentrifuge</td>
<td>Beckman Coulter</td>
<td>A95765</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ultracentrifuge Rotor 70.1 Ti</td>
<td>Beckman Coulter</td>
<td>342184</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Uranyl acetate</td>
<td>Agar Scientific Ltd</td>
<td>AGR1260A</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Whatman filter paper</td>
<td>Sigma-Aldrich</td>
<td>WHA10347510</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>&alpha;-Cyano-4-hydroxycinnamic acid</td>
<td>Sigma-Aldrich</td>
<td>C2020-25G</td>
<td>&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 ...

This protocol makes it possible to guarantee a good isolation of extracellular vesicles and notification of a higher number of EBDF proteins. Compared to other isolation techniques, this one maintains that it is more integrity and vertical activities. EVs are being studied more and more in LC as well as pathological conditions.This method can be applied to any systems that we choose to characterize and study the EVs. For that content or the biological ethics special attention is required when iterating EVs, as in the culture results in the following experiments. A visual demonstration is critical to host specific equipment such as a homemade size exclusion chromatography column as well as the involvement of nanoparticle counter and mass spectrometer.Helping to demonstrate the procedure will be Antonella Raffo-Romero, a from the laboratory. Start by pre-isolating extracellular vesicles, or EVs, from condition medium. Transfer the condition to culture medium from microglia or macrophage cultures into a conical tube and centrifuge it at 1, 200 times G for 10 minutes to pellet the cells.Transfer the supernatant into a new conical tube and centrifuge for another 20 minutes to eliminate apoptotic bodies. Then transfer the supernatant into a 10.4 milliliter polycarbonate tube. Place the tube into a 70.1 TI rotor and ultra centrifuge at at 100, 000 times G for 90 minutes at four degrees Celsius to pellet the EVs.After centrifugation, discard the supernatant and resuspend the pellet in 200 microliters of 0.2 micrometer filtered PBS. To isolate the EVs, prepare a homemade size exclusion chromatography column by washing and sterilizing a glass chromatography column, and placing a 60 microliter filter at the bottom. Stack the column with cross-linked agarose gel filtration base matrix to create a stationary phase of 0.6 centimeters in diameter and 20 centimeters in height.Then rinse the phase with 50 milliliters of 0.2 micro meter filtered PBS. If necessary, store the column at four degrees Celsius for later use. Place the resuspended EV pellet on top of the stationary phase and collect 20 sequential fractions of 250 microliters while continuing to add PBS to the top of the stationary phase.The fractions can be stored at minus 20 degrees Celsius. To perform a nanoparticle tracking analysis, make a dilution of each fraction with 0.2 micrometer filtered PBS and vortex it to eliminate aggregates. Put the solution in a one milliliter syringe and place it in the automated syringe pump.Next, adjust the camera settings to the appropriate screen gain level and camera level and click on Run. Load the sample in the analysis chamber with an infusion rate of 1000 for 15 seconds. Then decrease and stabilize the speed flow for video recording to an infusion rate of 25 for 15 seconds.Capture a three consecutive 60 second videos of the particle flow. Then adjust the camera level and the detection threshold before video analysis. Click on Settings OK to start the analysis, and click on Export when done.Wash the system with one milliliter of 0.2 micrometer filtered PBS between each fraction. If performing electron microscopy analysis, use a 50 kilodalton centrifugal filter to concentrate the size exclusion chromatography fractions of interest. For protein extraction, mix 50 microliters of RIPA buffer with the EV sample for five minutes on ice.Sonicate the samples three times at 500 Watts and 20 kilohertz for five seconds. Then remove vesicular debris by centrifuging at 20, 000 times G for 10 minutes at four degrees Celsius. After isolating the proteins, perform protein migration in the stacking gel of a 12%polyacrylic gel.Mix the proteins in the gel with Coomassie Blue for 20 minutes at room temperature. Then excise each colored gel piece and cut it into small pieces. Put the gel pieces through a series of successive washes as described in the manuscript.Then dry them completely with a vacuum concentrator. After drying perform protein reduction with 100 microliters of 100 millimolar ammonium bicarbonate containing 10 millimolar Dithiothreitol for one hour at 56 degrees Celsius. Next perform protein alkylation with 100 microliters of 100 millimolar ammonium bicarbonate containing 50 millimolar iodoacetamide for 45 minutes in the dark.Wash the gel pieces according to manuscript directions and completely dry them on the vacuum concentrator. Perform protein digestion with 50 microliters of trypsin in 20 millimolar ammonium bicarbonate at 37 degrees Celsius overnight. On the next day, extract the digested proteins from the gel with 50 microliters of 100%ACN for 30 minutes at 37 degrees Celsius and then 15 minutes at room temperature with continuous stirring.Then extract the proteins twice with 50 microliters of 5%TFA in 20 millimolar ammonium bicarbonate, while stirring continuously for 20 minutes. Add 100 microliters of ACN and continue stirring for 10 minutes. Afterwards, dry the proteins and resuspend them in 20 microliters of 0.1%TFA.Desalt the sample using a 10 microliter pipette tip with C 18 reverse phase media for desalting and concentrating peptides. Then elute them with ACN and 0.1%formic acid. Completely dry the sample with a vacuum concentrator and resuspend it in 20 microliters of ACN and 0.1%formic acid for liquid chromatography tandem mass spectrometry, or LC-MS.Load the digested peptides into the instrument and perform a sample analysis. To confirm EV isolation, each SCC fraction was subjected to a nanoparticle tracking analysis. The particle number was significantly higher in fractions five, six, and seven.These fractions were pooled into one sample 2F-EV positive, and compared with EV negative fractions, 1F-EV negative and 3F-EV negative, using Western blot analysis. The results showed the presence of heat shock protein 90 in the EV positive sample and in the cell lysate control. Electron microscopy of the EV positive sample showed EVs in a size range around 100 nanometers and 400 nanometers.Proteomics analysis was performed in order to identify contaminant proteins in EV negative samples and characterize the protein contents of EVs. The identified proteins were compared between 1F-EV negative and a 2F-EV positive samples, as well as between 2F-EV positive and a 3F-EV negative samples. Using this non-targeted method, a pool of 536 proteins were submitted to the ExoCarta database and led to the identification of 86 EV associated proteins.In addition, this pool also allowed for the identifying of protein interactions and their associated biological functions. It was found that the proteins from the EV positive sample played roles in immune and neuroprotective pathways. The effects of microglia-derived EVs were evaluated on neurite outgrowth with PC 12 cells and rat primary neurons.Significant outgrowth increase was observed under EVs compared to control. Macrophage-derived EVs were evaluated on glioma cell invasion. It was found that EVs impaired the growth and invasion of glioma spheroids.The most important thing to remember is to really carefully discard the supernatant of the ultracentrifugation step in order to prevent the loss of EVs in this procedure. We favor a large scale and non-targeted approach to focus on the whole protein content and then the vertical effect of EVs. so contents in nucleic acids can be associated using analysis.With this approach from a primary culture, we are able to discriminate between EV proteins and free proteins that are outside of variety. So the question remains whether this coagulation is artifactual or rather some other real

Research

JoVE Journal

Neuroscience

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Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

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