We are grateful to Rafael Prados-Rosales for sharing the anti-MEV antisera and Navneet Dogra for performing nanoparticle tracking analysis.

1. Preparation of Iron-depleted Defined Medium
<ol>
	<li>Prepare 1 L of minimal medium (MM) by dissolving 5 g of KH2PO4, 5 g of L-asparagine, 20 mL of glycerol, and 2 g of dextrose in 900 mL of deionized water in a plastic container. Avoid glass to prevent iron contamination. Adjust the pH to 6.8 with 5 N NaOH and the volume to 1 L with water.</li>
	<li>Add 50 g of metal chelating resin (MCR) and gently agitate using a magnetic stir bar for 24 h at 4 &#176;C. Sterilize and remove the MCR by filtration through a 0.22 &#181;m filter unit with a plastic receiver. To accelerate filtration and prevent filter clogging, let the resin sediment for about 30 min before filtration. 
	NOTE: This medium contains less than 2 &#181;M residual iron, as determined by atomic absorption spectroscopy.</li>
	<li>Supplement MM with 0.5 mg/L of ZnCl2, 40 mg/L of MgSO4, and 0.1 mg/L of MnSO4. Separately, prepare concentrated stocks (1,000x) of each of the metal supplements in deionized water and sterilize by filtration before MM supplementation. Iron-depleted, metal supplemented MM will be referred here as low iron MM (LIMM).</li>
	<li>From a 50 mM stock of FeCl3 dissolved in 10 mM HCl, add 1 mL to 1 L of LIMM (50 &#181;M final concentration) to prepare high iron MM (HIMM).</li>
</ol>
2. Growing Mycobacteria in Iron-limited Conditions
<ol>
	<li>Thaw out 50 &#181;L of a frozen 15% glycerol stock of Mtb and streak an agar plate supplemented with 10% ADN enrichment (5 g/L albumin, 2 g/L dextrose, and 0.85 g/L sodium chloride, 0.2% glycerol and 0.05% Tween-80)10. Incubate the plate at 37 &#176;C until colonies are visible.</li>
	<li>Inoculate a single colony of Mtb10 in 2 mL of mycobacterial broth medium (Table of Materials) supplemented with ADN enrichment. Incubate with agitation at 37 &#176;C.</li>
	<li>Let the Mtb culture grow to the late logarithmic phase (OD540&#160;is ~0.8). To check this, measure the OD540 using a spectrophotometer.</li>
	<li>Spread 200 &#181;L of the late logarithmic culture onto the mycobacterial agar plates (Table of Materials) supplemented with 0.2% glycerol, 0.05% Tween-80, and ADN. Inoculate at least 5 plates. Incubate the plates at 37 &#176;C until bacterial growth is visible as a confluent layer. This takes ~1 week for Mtb.</li>
	<li>Wet a sterile cotton swab in LIMM. Use this swab to collect bacteria from the agar plates and inoculate 100 mL of LIMM to prepare a concentrated bacterial suspension with an OD540 of ~1.0.</li>
	<li>Dilute this suspension 10 times to 1 L with LIMM and divide it into two, 2 L sterile plastic bottles, each one containing 500 mL of culture.</li>
	<li>Take out 2 mL of culture and transfer it to a 5 mL culture tube. Add 10 &#181;L of 10% vol/vol tyloxapol.</li>
	<li>Incubate the cultures at 37 &#176;C standing for 14 days.</li>
</ol>
3. Collection of MEVs
<ol>
	<li>Measure the OD540 of the 2 mL culture at the time of collection. Make 1:10 serial dilutions of the culture and plate 100 &#181;L of each dilution on agar plates with ADN and 0.05% Tween-80.</li>
	<li>Transfer the culture to five 225 mL conical centrifuge tubes and centrifuge at 2,850 x g for 7 min at 20 &#176;C.</li>
	<li>Collect the culture supernatant with a 50 mL pipette and filter sterilize it through a 0.22 &#181;m filter unit.</li>
</ol>
4. Isolation of MEVs
<ol>
	<li>Transfer the culture filtrate into a stirred cell ultrafiltration system placed at 4 &#176;C and filter the concentrate at &#60;50 psi through a 100 kDa cutoff membrane to ~50 mL.</li>
	<li>Centrifuge the concentrated culture filtrate at 15,000 x g for 15 min at 4 &#176;C and collect the supernatant.</li>
	<li>Centrifuge the culture filtrate in polycarbonate ultracentrifugation tubes at 100,000 x g for 2 h at 4 &#176;C.</li>
	<li>Resuspend the membranous pellets in a total of 1 mL of sterile phosphate buffered saline (PBS) by gentle pipetting.</li>
	<li>Mix 0.5 mL of the pellet suspension obtained in step 4.4 with 1.5 mL of 60% iodixanol solution, yielding a final iodixanol concentration of 45% wt/vol. Dispense this mix at the bottom of a 13 mm x 51 mm polypropylene thin-walled ultracentrifuge tube.</li>
	<li>Overlay the MEV-iodixanol 45% suspension with 1 mL of 40%, 35%, 30%, 25%, and 20% (vol/vol in PBS) iodixanol solutions and 1 mL of PBS at the top.</li>
	<li>Centrifuge at 100,000 x g for 18 h at 4 &#176;C.</li>
	<li>Collect the 1 mL density gradient fractions starting from the top using a 1 mL Hamilton syringe.</li>
	<li>Dilute each collected fraction to 20 mL with PBS and centrifuge at 100,000 x g for 2 h at 4 &#176;C.</li>
	<li>Remove the supernatant and suspend the pellet in 0.5 mL of PBS. Store this pellet at 4 &#176;C.</li>
</ol>
5. Quantification of MEVs
<ol>
	<li>Measure the protein concentration in each fraction by a Bradford assay (Table of Materials), following the manufacturer&#39;s guidelines.</li>
	<li>Perform membrane lipid analysis.
	<ol>
		<li>Incubate 10 &#181;L of each gradient fraction with the fluorescent membrane probe 1-(4-Trimethylammoniumphenyl)-6-Phenyl-1,3,5-Hexatriene p-Toluenesulfonate (TMA-DPH) at a final concentration of 1 &#181;g/mL in a final 50 &#181;L volume of PBS in 96 well black plates.</li>
		<li>Incubate the plates at 33 &#176;C for 20 min.</li>
		<li>Measure the fluorescence at 360 nm excitation and 430 nm emission.</li>
	</ol>
	</li>
</ol>
6. Qualitative analysis of MEVs
<ol>
	<li>Perform protein electrophoresis.
	<ol>
		<li>Mix approximately 1 &#181;g of MEV samples in 16 &#181;L with 4 &#181;L of 5x sample loading buffer (10% w/v SDS, 10 mM dithiothreitol, 20% v/v glycerol 0.2 M Tris-HCl, pH 6.8 0.05% w/v bromophenol blue) and heat the samples in sample buffer at 85 &#176;C for 5 min.</li>
		<li>Load in a 10% Tris/Glycine SDS-polyacrylamide gel11 and run at 10 V/cm in running buffer (25 mM Tris base, 190 mM glycine, 0.1% SDS) until the blue dye front reaches the bottom of the gel.</li>
		<li>Stain the gel with an ultrasensitive protein staining solution (Table of Materials).</li>
	</ol>
	</li>
	<li>Perform the dot blot.
	<ol>
		<li>Load 2 &#181;L of a MEVs suspension with a concentration of approximately 0.5 &#181;g/&#181;L, and twofold serial dilutions on a nitrocellulose membrane and process for a dot blot according to the manufacturer&#39;s instructions.</li>
		<li>Use an antiserum raised in mice against a preparation of MEVs at a dilution of 1:5,000 as the primary antibody and a goat anti-mouse coupled to horseradish peroxidase (HRP) at a 1:10,000 dilution as the secondary antibody. Detect antigen-antibody complexes with an appropriate HRP substrate Blotting Detection Reagent and an imaging system.</li>
	</ol>
	</li>
	<li>Perform negative staining and electron microscopy.
	<ol>
		<li>Fix 250 &#181;L of MEVs with 2% glutaraldehyde in 0.1 M cacodylate at room temperature for 2 h and incubate overnight in 4% formaldehyde, 1% glutaraldehyde, and 0.1% PBS.</li>
		<li>Stain the fixed samples with 2% osmium tetroxide for 90 min.</li>
		<li>Serially dehydrate the sample in ethanol and embed in Spurr&#39;s epoxy resin.</li>
		<li>Observe the MEVs under a transmission electron microscope.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Prados-Rosales, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacteria+release+active+membrane+vesicles+that+modulate+immune+responses+in+a+TLR2-dependent+manner+in+mice.">Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice.</a> Journal of Clinical Investigation. 121, 1471-1483 (2011).</li><li>Gupta, S., Rodriguez, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterial+extracellular+vesicles+and+host+pathogen+interactions.">Mycobacterial extracellular vesicles and host pathogen interactions.</a> Pathogens and Disease. 76 (4), (2018).</li><li>Athman, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Membrane+Vesicles+Mediate+the+Release+of+Mycobacterium+tuberculosis+Lipoglycans+and+Lipoproteins+from+Infected+Macrophages.">Bacterial Membrane Vesicles Mediate the Release of Mycobacterium tuberculosis Lipoglycans and Lipoproteins from Infected Macrophages.</a> Journal of Immunology. 195, 1044-1053 (2015).</li><li>Athman, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+tuberculosis+Membrane+Vesicles+Inhibit+T+Cell+Activation.">Mycobacterium tuberculosis Membrane Vesicles Inhibit T Cell Activation.</a> Journal of Immunology. 198, 2028-2037 (2017).</li><li>Rath, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+regulation+of+vesiculogenesis+and+immunomodulation+in+Mycobacterium+tuberculosis.">Genetic regulation of vesiculogenesis and immunomodulation in Mycobacterium tuberculosis.</a> Proceedings of the National Academy of Science U.S.A. 110, E4790-E4797 (2013).</li><li>White, D. W., Elliott, S. R., Odean, E., Bemis, L. T., Tischler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+tuberculosis+Pst/SenX3-RegX3+Regulates+Membrane+Vesicle+Production+Independently+of+ESX-5+Activity.">Mycobacterium tuberculosis Pst/SenX3-RegX3 Regulates Membrane Vesicle Production Independently of ESX-5 Activity.</a> mBio. 9, pii 00778 (2018).</li><li>Dauros Singorenko, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+membrane+vesicles+from+prokaryotes:+a+technical+and+biological+comparison+reveals+heterogeneity.">Isolation of membrane vesicles from prokaryotes: a technical and biological comparison reveals heterogeneity.</a> Journal of Extracellular Vesicles. 6, 1324731 (2017).</li><li>Prados-Rosales, R., Brown, L., Casadevall, A., Montalvo-Quiros, S., Luque-Garcia, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+identification+of+membrane+vesicle-associated+proteins+in+Gram-positive+bacteria+and+mycobacteria.">Isolation and identification of membrane vesicle-associated proteins in Gram-positive bacteria and mycobacteria.</a> MethodsX. 1, 124-129 (2014).</li><li>Prados-Rosales, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+Mycobacterium+tuberculosis+membrane+vesicles+in+iron+acquistion.">Role for Mycobacterium tuberculosis membrane vesicles in iron acquistion.</a> Journal of Bacteriology. 196, 1250-1256 (2014).</li><li>Sanders, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+Laboratory+Techniques:+Plating+Methods.">Aseptic Laboratory Techniques: Plating Methods.</a> Journal of Visualized Experiments. 63, e3064 (2012).</li><li>Harlow, E., Lane, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Antibodies. 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The authors have no conflicts of interest.

Multiple methods to purify eukaryotic cell-derived exosomes have been developed12. In contrast, there is limited information on effective methods to purify bacteria-derived EVs7. Efficient isolation of Mtb-derived EVs needs to consider the intrinsic difficulties in growing this pathogenic mycobacterium. Mtb has a long division time (~24 h) and should be handled in biosafety level three (BSL-3) conditions. Therefore, it is important to optimize the efficiency of MEV isolation methods. Because mycobacteria release glycolipids and other hydrophobic molecules that aggregate and easily contaminate crude MEV preparations into the medium, it is important to purify and validate MEVs before conducting biochemical and functional studies. Based on previous observations that demonstrated that Mtb enhances the release of MEVs under conditions of iron limitation, a protocol was established for EV purification from iron-limited mycobacteria. It has also been confirmed that non-virulent M. smegmatis also increases release of EVs in response to low iron conditions (data not shown). Therefore, the same protocol could be used to purify EVs from this bacterium in BSL-2 conditions.
A critical step of this procedure is the preparation of the low iron medium. This medium should be prepared as described here and stored in a plastic container, not in glass, to prevent iron contamination. Supplements commonly used in Mtb growth medium to stimulate bacterial growth and prevent characteristic mycobacterial clumping, such as bovine serum albumin, Tween-80, or tyloxapol, must be avoided. These additives lead to lipoprotein complex artifacts that copurify with vesicles and reduce vesicle yield. For CFU determination, a small culture in medium supplemented with detergent (Tween-80 or tyloxapol) can be set in parallel to the detergent-free large culture. MEVs in the culture filtrate are stable at 4 &#176;C for several days. Therefore, if not processed immediately, the culture filtrate can be stored refrigerated.
The total yield of purified MEV from 1 L of culture was around 500 &#181;g/L protein, which is sufficient to conduct multiple analyses such as proteomics, lipidomics, and functional assays. Depending on the type of assay, sufficient MEVs can be isolated from smaller volumes (i.e., 250 mL). This facilitates comparative analysis of conditions and factors influencing MEV release.
This is an effective method to purify MEVs, but it has limitations. It is a long procedure with multiple ultracentrifugation steps. In the future, this method will be compared to gel filtration chromatography, and as molecular markers of MEVs are discovered, affinity capture methods could be implemented. The host environment is iron-limited, therefore MEVs produced by Mtb in a low iron medium are probably more closely related to MEVs produced during infection and could provide relevant insights about the role of MEVs in tuberculosis pathogenesis.

Mycobacterial extracellular vesicles (MEVs) are membrane-bound nanoparticles, 60&#8722;300 nm in size, naturally released by fast- and slow-growing mycobacteria1. MEVs released by pathogenic mycobacteria constitute a mechanism to interact with the host via immunologically active proteins, lipids, and glycolipids secreted in a concentrated and protected manner2,3,4. To characterize MEVs and understand their biogenesis and functions, strict and efficient methods of vesicle purification and validation are crucial. Thus far, MEVs have been isolated from the culture filtrates of mycobacteria grown in an iron-rich medium1,5,6,7,8.
However, previous work demonstrated that iron limitation greatly stimulates vesicle release in Mtb,possibly to capture iron via mycobactin, a siderophore secreted in MEVs9. Although procedures for MEVs isolation from Mtb cultured in high iron medium have been described, an efficient methodology to obtain MEVs from low iron cultures has not been reported. Therefore, the goal of this method is to isolate, purify, and quantify MEVs obtained from low iron cultures so that they can be used for biochemical and functional assays and for the analysis of genetic determinants of vesicle production in mycobacteria.

<table><tbody><tr><td>Amicon stirred cell Model 108</td><td>EMD Milipore</td><td>UFSC40001</td><td>Cell Ultrafiltration system</td></tr><tr><td>BD Polypropilene 225 mL conical tubes</td><td>Fisher</td><td>05-538-61</td><td>Conical centrifuge tubes</td></tr><tr><td>Biomax 100 kDa cut-off ultrafiltration membrane</td><td>EMD Milipore</td><td>PBHK07610</td><td>Ultrafiltration membrane</td></tr><tr><td>Chelex-100 resin</td><td>Bio-Rad</td><td>142-2842</td><td>Metal chelating resin</td></tr><tr><td>Middlebrook 7H10 Agar</td><td>BD Difco</td><td>262710</td><td>Mycobacterial Agar plates</td></tr><tr><td>Middlebrook 7H9 Broth</td><td>BD Difco</td><td>271310</td><td>Mycobacterial broth medium</td></tr><tr><td>Nitro cellulose blotting membrane</td><td>GE Healthcare</td><td>10600001</td><td>Blotting Membrane</td></tr><tr><td>Optiprep</td><td>Sigma</td><td>D1556</td><td>Iodixanol</td></tr><tr><td>Polycarbonate ultra centrifugation tubes 25 mm x 89 mm</td><td>Beckman Coulter</td><td>355618</td><td>Polycarbonate ultra centrifugation tubes 25 mm x 89 mm</td></tr><tr><td>Polypropylene thin walled centrifuge tube 13 mm x 15 mm</td><td>Beckman Coulter</td><td>344059</td><td>Polypropylene thin walled centrifuge tube 13 mm x 15 mm</td></tr><tr><td>Protein Assay dye</td><td>BioRad</td><td>5000006</td><td>Bradford Protein Staining</td></tr><tr><td>SYPRO Ruby</td><td>Molecular Probes</td><td>S12000</td><td>Ultrasensitive protein stain</td></tr><tr><td>TMA-DPH</td><td>Molecular Probes</td><td>T204</td><td>1-(4-Trimethylammoniumphenyl)-6-Phenyl-1,3,5-Hexatriene &lt;em&gt;p&lt;/em&gt;-Toluenesulfonate</td></tr><tr><td>Vacuum filtration flasks</td><td>CellPro</td><td>V50022</td><td>Filter Unit</td></tr></tbody></table>

isolation and characterization of extracellular vesicles produced by iron-limited mycobacteria

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) containing immunologically active molecules. Knowledge regarding the molecular mechanisms of vesicle biogenesis, the content of the vesicles, and their functions at the pathogen-host interface is very limited. Addressing these questions requires rigorous procedures for isolation, purification, and validation of EVs. Previously, vesicle production was found to be enhanced when M. tuberculosis was exposed to iron restriction, a condition encountered by Mtb in the host environment. Presented here is a complete and detailed protocol to isolate and purify EVs from iron-deficient mycobacteria. Quantitative and qualitative methods are applied to validate purified EVs.

MEVs were purified by differential sedimentation in a density gradient (Figure 1, Figure 2). Under the conditions described, MEVs separated mostly in gradient fraction 3 (F3), which corresponds to 25% iodixanol. This conclusion is based on the detection of protein, membrane lipid, microscopic visualization of intact MEVs, nanoparticle size distribution, and positive reactivity with an antivesicle antiserum (Figure 2, Figure 3). Protein and lipid concentration normalized to colony-forming units (CFUs) showed an approximately eightfold increase of MEV yield in low iron relative to high iron conditions (50 &#181;M FeCl3) (Figure 3). Although the results of one representative experiment is presented, this is a highly reproducible result based on multiple (&#62;10) isolations of MEVs. The pure MEV yield obtained from a 1 L low iron culture by this method was approximately 500 &#181;g of protein.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60359/60359fig1.jpg" /> 
Figure 1: Diagrammatic representation of the methodology used for MEV purification and quantification. Mycobacteria grown in agar plates were used to inoculate iron-depleted minimal medium and grow Mtb for EV isolation. MEVs were purified by a discontinuous density gradient from the cell-free culture filtrate. A combination of membrane lipid and vesicle protein determination, microscopy, and nanoparticle analysis was implemented to characterize purified MEVs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60359/60359fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60359/60359fig2.jpg" /> 
Figure 2: Characterization of purified MEVs. (A) Shown are photographs of an actual density gradient separation of crude MEVs and the pellet of purified MEVs collected by ultracentrifugation of gradient fraction 3 (F3). (B) SDS-gel stained showing the protein profile of the various density gradient fractions. (C) Dot blot analysis showing vesicle-associated proteins concentrated in F3. (D) MEVs present in F3 observed by negative staining. (E) MEV size distribution according to nanoparticle analysis (NTA). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60359/60359fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60359/60359fig3.jpg" /> 
Figure 3: Comparative analysis of MEV yield in low and high iron cultures. A representative result of (A) protein and lipid quantification and (B) dot blot analysis of purified MEVs isolated from iron-limited and iron sufficient Mtb cultures. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60359/60359fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria at JoVE.com

Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

Mycobacterium tuberculosis shows increased production and release of extracellular vesicles in response to low iron conditions. This work details a protocol for generating low iron conditions and methods for the purification and characterization of mycobacterial extracellular vesicles released in response to iron deficiency.

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) ...

isolation-characterization-extracellular-vesicles-produced-iron

Nanyang Technological University

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JoVE Journal

Immunology and Infection

7.2K Views.  Rutgers, The State University of New Jersey. Mycobacterium tuberculosis shows increased production and release of extracellular vesicles in response to low iron conditions. This work details a protocol for generating low iron conditions and methods for the purification and characterization of mycobacterial extracellular vesicles released in response to iron deficiency.

Video: Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

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 ...

Neuroscience

Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography

The role of extracellular vesicles (EVs) in the context of bacterial infection has emerged as a new avenue for understanding microbial physiology. Specifically, Mycobacterium tuberculosis (Mtb) EVs play a role in the host-pathogen interaction and response to environmental stress. Mtb EVs are also highly antigenic and show potential as vaccine components. The most common method for purifying Mtb EVs is density gradient ultracentrifugation. This process has several limitations, including low throughput, low yield, reliance on expensive equipment, technical challenges, and it can negatively impact the resulting preparation. Size exclusion chromatography (SEC) is a gentler alternative method that combats many of the limitations of ultracentrifugation. This protocol demonstrates that SEC is effective for Mtb EV enrichment and produces high-quality Mtb EV preparations of increased yield in a rapid and scalable manner. Additionally, a comparison to density gradient ultracentrifugation by quantification and qualification procedures demonstrates the benefits of SEC. While the evaluation of EV quantity (nanoparticle tracking analysis), phenotype (transmission electron microscopy), and content (Western blotting) is tailored to Mtb EVs, the workflow provided can be applied to other mycobacteria.

Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography

This protocol describes size exclusion chromatography, a facile and reproducible technique for enriching Mycobacterium tuberculosis extracellular vesicles from culture supernatants.

The role of extracellular vesicles (EVs) in the context of bacterial infection has emerged as a new avenue for understanding microbial physiology. ...

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 ...

Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, including metabolites, lipids, proteins, and nucleic acids, several groups have evaluated either the native or recombinant versions of bEVs for their protective potency as subunit vaccine candidates. Unlike native EVs, recombinant EVs are molecularly engineered to contain one or more immunogens of interest. Over the last decade, different groups have explored diverse approaches for generating recombinant bEVs. However, here, we report the design, construction, and enrichment of recombinant mycobacterial EVs (mEVs) in mycobacteria. Towards that, we use Mycobacterium smegmatis (Msm), an avirulent soil mycobacterium as the model system. We first describe the generation and enrichment of native EVs of Msm. Then, we describe the design and construction of recombinant mEVs that contain either mCherry, a red fluorescent reporter protein, or EsxA (Esat-6), a prominent immunogen of Mycobacterium tuberculosis. We achieve this by separately fusing mCherry and EsxA N-termini with the C-terminus of a small Msm protein Cfp-29. Cfp-29 is one of the few abundantly present proteins of MsmEVs. The protocol to generate and enrich recombinant mEVs from Msm remains identical to the generation and enrichment of native EVs of Msm.

This protocol details the enrichment of native mycobacterial extracellular vesicles (mEVs) from axenic cultures of Mycobacterium smegmatis (Msm) and how mCherry (a red fluorescent reporter)-containing recombinant MsmEVs can be designed and enriched. Lastly, it verifies the novel approach with the enrichment of MsmEVs containing the EsxA protein of Mycobacterium tuberculosis.

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, ...

Isolation and Characterization of Microvesicles from Peripheral Blood

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived microvesicles (MVs, diameter &#62; 100 nm) is a fundamental cellular process that occurs in all living cells. These vesicles transport proteins, lipids and nucleic acids specific for their cell of origin and in vitro studies have highlighted their importance as mediators of intercellular communication. EVs have been successfully isolated from various body fluids and especially EVs in blood have been identified as promising biomarkers for cancer or infectious diseases. In order to allow the study of MV subpopulations in blood, we present a protocol for the standardized isolation and characterization of MVs from peripheral blood samples. MVs are pelleted from EDTA-anticoagulated plasma samples by differential centrifugation and typically possess a diameter of 100 - 600 nm. Due to their larger size, they can easily be studied by flow cytometry, a technique that is routinely used in clinical diagnostics and available in most laboratories. Several examples for quality control assays of the isolated MVs will be given and markers that can be used for the discrimination of different MV subpopulations in blood will be presented.

Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting.

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

Biology

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 Characterization of Cyanobacterial Extracellular Vesicles

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential biotechnology models. Recent work has demonstrated that both marine and freshwater cyanobacteria produce extracellular vesicles -&#160;small membrane-bound structures released from the outer surface of the microbes. While vesicles likely contribute to diverse biological processes, their specific functional roles in cyanobacterial biology remain largely unknown. To encourage and advance research in this area, a detailed protocol is presented for isolating, concentrating, and purifying cyanobacterial extracellular vesicles. The current work discusses methodologies that have successfully isolated vesicles from large cultures of Prochlorococcus, Synechococcus, and Synechocystis. Methods for quantifying and characterizing vesicle samples from these strains are presented. Approaches for isolating vesicles from aquatic field samples are also described. Finally, typical challenges encountered with cyanobacterial vesicle purification, methodological considerations for different downstream applications, and the trade-offs between approaches are also discussed.

The present protocol providesdetailed descriptions for isolation, concentration,and characterization of extracellular vesicles from cyanobacterial cultures. Approaches for purifying vesicles from cultures at different scales, trade-offs among methodologies, and considerations for working with field samples are also discussed.

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential ...

Bacterial Culture Filtrate Protein Concentration using an Ultrafiltration Device

Source: Ryan, J. M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63895">Mycobacterium tuberculosis</a><a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63895"> Extracellular Vesicle Enrichment through Size Exclusion Chromatography.</a> J. Vis. Exp. (183) (2022)
This video demonstrates the isolation and concentration of bacterial culture filtrate proteins, which include bacterial extracellular vesicles, using ultrafiltration. The concentrated extracellular vesicles serve as diagnostic markers for disease detection.

Zagęszczenie białka filtratu kultur bakteryjnych za pomocą urządzenia do ultrafiltracji

1. Preparation of crude Mycobacterium tuberculosis (Mtb) extracellular vesicle (EV) concentrate
NOTE: For detailed procedures on the cultivation of Mtb ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

In Vitro and In Vivo Models

A Size Exclusion Chromatography Technique to Purify Mycobacterial Extracellular Vesicles

Source: Ryan, J. M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63895">Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography.</a> J. Vis. Exp. (2022).
This video demonstrates the size exclusion chromatography (SEC) technique for purifying bacterial extracellular vesicles (EVs) from the culture filtrate of Mycobacterium tuberculosis (Mtb). The technique utilizes a porous resin-packed column to obtain purified fractions of EVs. The porous particles selectively allow smaller proteins to enter the pores, resulting in slower elution. At the same time, EVs are excluded from entering the pores due to their larger size and are eluted first.

Technika chromatografii wykluczającej wielkość do oczyszczania pęcherzyków zewnątrzkomórkowych prątków

1. Preparation of crude Mtb EV concentrate
NOTE: For detailed procedures on the cultivation of Mtb and preparation of culture filtrate protein (CFP), see ...

SEC-based Isolation of Microglia-derived Extracellular Vesicles: A Technique to Isolate Extracellular Vesicles from Microglia Conditioned Culture Media

Transfer the conditioned culture medium from microglia or macrophage cultures into a conical tube and centrifuge it at 1,200 x 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 ultracentrifuge it at 100,000 x g for 90 minutes at 4 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 home-made size exclusion chromatography column by washing and sterilizing a glass chromatography column and placing a 60-micrometer 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-micrometer filtered PBS. If necessary, store the column at 4 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.

Izolacja pęcherzyków zewnątrzkomórkowych pochodzących z mikrogleju oparta na SEC: technika izolowania pęcherzyków zewnątrzkomórkowych z pożywek hodowlanych kondycjonowanych mikroglejem

Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

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

Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography

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

Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria

Isolation and Characterization of Microvesicles from Peripheral Blood

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

Isolation and Characterization of Cyanobacterial Extracellular Vesicles

Zagęszczenie białka filtratu kultur bakteryjnych za pomocą urządzenia do ultrafiltracji

Technika chromatografii wykluczającej wielkość do oczyszczania pęcherzyków zewnątrzkomórkowych prątków

Izolacja pęcherzyków zewnątrzkomórkowych pochodzących z mikrogleju oparta na SEC: technika izolowania pęcherzyków zewnątrzkomórkowych z pożywek hodowlanych kondycjonowanych mikroglejem