This work was funded by the National Science Foundation (1554417) and National Institutes of Health (DE027769).

1. Preparation of buffers
<ol>
	<li>To prepare the ELISA wash buffer, add 3.94 g Tris-base, 8.77 g NaCl, and 1 g bovine serum albumin (BSA) to 1 L of deionized (DI) water. Add 500 &#181;L polysorbate-20. Adjust the pH to 7.2 using HCl or NaOH.</li>
	<li>To prepare the blocking buffer, add 3.94 g Tris-base, 8.77 g NaCl, and 10 g BSA. Add 500 &#181;L polysorbate-20 to 1 L of DI water. Adjust the pH to 7.2 using HCl or NaOH.</li>
	<li>To prepare the elution buffer (PBS), add 8.01 g NaCl, 2.7 g KCl, 1.42 g Na2HPO4, and 0.24 g KH2PO4 to 1 L DI water. Adjust the pH to 7.4 using HCl or NaOH. 
	&#8203;NOTE: A 10x solution of this buffer can be made and diluted with DI water as needed.</li>
</ol>
2. Preparation of OMV sample
<ol>
	<li>Grow A. actinomycetemcomitans cells to the late exponential phase (optical density at 600 nm of 0.7). Pellet the cells by centrifuging twice at 10,000 x g at 4 &#176;C for 10 min. Filter the supernatant through a 0.45 &#181;m filter.</li>
	<li>Concentrate the bacteria-free supernatant using 50 kDa-molecular weight cut-off filters. Ultracentrifuge the concentrated solution at 105,000 x g at 4 &#176;C for 30 min.&#160;</li>
	<li>Resuspend the pellet in PBS and ultracentrifuge again (105,000 x g at 4 &#176;C for 30 min.) Resuspend the pellet in 2 mL of PBS.</li>
</ol>
3. Packing the S-1000 column
<ol>
	<li>Mix the stock bottle of gel filtration medium with a glass stir rod and pour out into a glass bottle the volume necessary to fill the column, plus approximately 50% excess (about 135 mL). Let these beads sit until they have settled, and then decant off the excess liquid. Resuspend the beads in elution buffer, so that the final solution is approximately 70% (by volume) gel, 30% buffer. Degas the solution under vacuum.</li>
	<li>Mount the glass column vertically using a ring stand and fill with elution buffer to wet the walls of the column. Drain the buffer until there is only about 1 cm of buffer remaining in the column.</li>
	<li>Without creating bubbles, carefully pipette beads into the column, filling the column to the top. Continue to drain excess buffer throughout this process. Be sure to not let the beads settle completely before adding additional beads to the top of the column. The column should be packed to a height of about 2 cm below the bottom of the column reservoir.</li>
</ol>
4. Loading the sample and collecting fractions
<ol>
	<li>Degas the elution buffer under vacuum. Wash the column with two column-volumes (180 mL) of elution buffer.</li>
	<li>Allow the remaining buffer to fully enter the column. Once the buffer has reached the top of the gel layer, carefully pipette a 2-mL sample containing OMVs (at a lipid concentration of approximately 100 - 200 nmol/L) onto the surface of the beads, being careful not to disturb any of the beads at the top of the column. Allow the sample to fully enter the gel, that is, when no liquid remains above the gel layer.&#160;</li>
	<li>Carefully and slowly add elution buffer on top of the gel column. Do not disturb the top layer of the gel, as this will cause sample dilution.</li>
	<li>Place a single 50-mL tube under the column and open the column. Collect the first 20 mL of the eluent. Add additional elution buffer to the top of the column, carefully, as needed to ensure the column is never dry.</li>
	<li>Place a series of 1.5 mL tubes under the column. Start the column and collect a series of 1-mL samples in each tube. As the samples are being collected, continue to add elution buffer to the top of the column, as necessary. Repeat until 96 fractions have been collected. Stop the column. 
	NOTE: The samples should be stored at -20 &#176;C for long-term storage or 4 &#176;C for short-term storage until further analysis.</li>
	<li>To clean the column, run one column volume (90 mL) of 0.1 M NaOH through the column. Run two column volumes (180 mL) of elution buffer through the column.</li>
</ol>
5. Sample analysis
<ol>
	<li>To measure the lipid concentration in each fraction, pipette 50 &#181;L of each fraction into a single well of a 96-well plate. To each well, add 2.5 &#181;L of lipophilic dye. Incubate for 15 s. Measure the fluorescence intensity on a plate reader with an excitation wavelength of 515 nm and an emission wavelength of 640 nm. To calculate the fraction of all lipid in each sample, sum all of the emission intensities and divide each individual intensity by the total.</li>
	<li>To measure the concentration of a particular protein, pipette 100 &#181;L of each fraction into a single well of an ELISA immuno-plate. Incubate at 25 &#176;C for 3 h.
	<ol>
		<li>Decant the samples. Add 200 &#181;L of ELISA wash buffer to each well and decant. Repeat four times for a total of five washes.</li>
		<li>Add 200 &#181;L of blocking buffer to each well and incubate for 1 h at 25 &#176;C. Decant.</li>
		<li>Incubate plates with 100 &#181;L blocking buffer plus primary antibody (1:10,000 for purified antibody; 1:10 for unpurified antibody) overnight at 4 &#176;C. Decant.</li>
		<li>Add 200 &#181;L of ELISA wash buffer to each well and decant. Repeat four times for a total of five washes.</li>
		<li>Add 100 &#181;L of ELISA wash buffer plus secondary antibody (1:30,000) to each well. Incubate for 1 h at 25 &#176;C.</li>
		<li>Add 200 &#181;L of ELISA wash buffer to each well and decant. Repeat four times for a total of five washes.</li>
		<li>Add 100 &#181;L of the 3,3&#39;,5,5&#39;-tetramethylbenzidine (TMB) one-step solution and incubate for 15-30 min or until a blue color develops. Stop the TMB reaction with 50 &#181;L of the stopping solution.</li>
		<li>On a plate reader, read the absorbance of each well at a wavelength of 450 nm.</li>
	</ol>
	</li>
	<li>To measure the total protein concentration, record the absorbance at a wavelength of 280 nm (A280) of each fraction, using a UV-vis spectrophotometer.</li>
</ol>
A schematic of the protocol is shown in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62429/62429fig01.jpg" /> 
Figure 1: Schematic of SEC procedure. The column is packed with degassed gel filtration medium carefully to avoid bubbles and discontinuities, then washed with two column volumes of elution buffer. Next, the sample is carefully pipetted onto the top of the gel, without disrupting gel packing. The column is opened and run until the sample completely enters the gel. At this point, buffer is placed on the top of the column, and the first 20 mL of eluate is collected. Next, a series of 1-mL fractions is collected. These fractions are then placed in a 96-well plate or 96-well immuno-plate for analysis of lipid and protein content. <a href="https://www.jove.com/files/ftp_upload/62429/62429fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have no conflicts of interest to report.

Here, we have provided a protocol for the simple, fast, and reproducible separation of bacterial OMV subpopulations. Although the technique is relatively straight-forward, there are some steps that must be performed extremely carefully to ensure that efficient separation occurs in the column. First, it is essential that the gel be loaded into the column carefully and slowly to avoid air bubbles. We have observed that leaving the gel at room temperature for several hours before loading the column allows the gel to equilibrate and minimizes bubble formation within the column. When the gel is pipetted into the column, it should be carefully pipetted along the side of the column to minimize turbulence. At all times during loading, excess buffer should be maintained in the column to avoid discontinuities in the settled gel. If a disjunction should occur, add more buffer and pipette up and down to resuspend the gel.
Similarly, loading the column with sample is critically important. Because the sample will become diluted as it passes through the column, the loaded sample should be sufficiently concentrated before separation by SEC. For the A. actinomycetemcomitans OMVs, we have found that a 1-mL sample containing approximately 100-200 nmol/L of lipids is ideal. After the sample is loaded carefully at the top of the column without disrupting the gel layer, the column should be run just until the sample fully enters the gel column. At this point, the column should be stopped so that a layer of buffer can be carefully added to the top of the gel. It is helpful to load only a small volume (~ 1 mL) of buffer, ensuring that the gel layer is not disrupted. Once the sample has been run further into the gel, buffer can be added in larger volumes and the concern with disrupting the gel layer is less of an issue. The column can be reused multiple times, as long as it is maintained in a fully hydrated state and cleaned well (Step 4.6) between runs. 
All OMV purification procedures follow the same initial steps that include bacterial growth, removal of bacterial cells, and OMV isolation27. Although this "crude" preparation has commonly been used in OMV studies28, it is becoming increasingly apparent that a subsequent purification step is necessary to remove co-precipitating proteins and other contaminants, as well as to separate OMV subpopulations. In OMV studies, this purification step is commonly completed using density gradient centrifugation. In the eukaryotic extracellular vesicle field, the use of SEC to separate vesicle populations and to remove contaminants is increasing in importance, as it is simpler, faster, and less expensive than DGC29. In addition, SEC has the advantage of being possible to automate, unlike DGC29. Thus, while DGC remains the "gold standard" of vesicle isolation in the bacterial OMV field, we propose that the numerous advantages of SEC make it an extremely useful, if not better, method of OMV purification than DGC. In this work, we have demonstrated that a 1.5 x 50 cm column of Sephacryl S-1000 is capable of separating two subpopulations of OMVs. We have also observed that the approach is capable of removing nucleic acids and free proteins from the OMV solution. Previous reports have found SEC to be able to remove free LPS from OMV preparations, as well28. 
In conclusion, we propose that SEC holds much promise in the purification of bacterial vesicles. While we have demonstrated the ability of the technique to separate subpopulations of OMVs produced by a specific bacterium (A. actinomycetemcomitans), we anticipate that the technique will be found to be extremely valuable in analyzing other bacterial vesicle samples, as it sees additional use. In particular, as the biotechnological applications of OMVs increase, the need for consistent and pure vesicle preparations will also increase; SEC is a promising method for these applications.

Gram-negative bacteria release vesicles derived from their outer membrane, so-called outer membrane vesicles (OMVs), throughout growth. These OMVs play important roles in cell-to-cell communication, both between bacteria and host as well as between bacterial cells, by carrying a number of important biomolecules, including DNA/RNA, proteins, lipids, and peptidoglycans1,2. In particular, the role of OMVs in bacterial pathogenesis has been extensively studied due to their enrichment in certain virulence factors and toxins3,4,5,6,7,8,9,10,11.
OMVs have been reported to range in size from 20 to 450 nm, depending on the parent bacteria and the growth stage, with several types of bacteria releasing heterogeneously sized OMVs8,12,13,14, which also differ in their protein composition and mechanism of host cell entry12. H. pylori released OMVs ranging in diameter from 20 to 450 nm, with the smaller OMVs containing a more homogeneous protein composition than the larger OMVs. Importantly, the two populations of OMVs were observed to be internalized by host cells via different mechanisms12. In addition, we have demonstrated that Aggregatibacter actinomycetemcomitans releases a population of small (&#60;150 nm) OMVs along with a population of large (&#62;350 nm) OMVs, with the OMVs containing a significant amount of a secreted protein toxin, leukotoxin (LtxA)15. While the role of OMV heterogeneity in cellular processes is clearly important, technical difficulties in separating and analyzing distinct populations of vesicles has limited these studies.
In addition to their importance in bacterial pathogenesis, OMVs have been proposed for use in a number of biotechnological applications, including as vaccines and drug delivery vehicles16,17,18,19,20. For their translational use in such approaches, a clean and monodisperse preparation of vesicles is required. Thus, effective&#160;and efficient methods of separation are necessary.
Most commonly, density gradient centrifugation (DGC) is used to separate heterogeneously sized vesicle populations from cellular debris, including flagellae and secreted proteins21; the method has also been reported as an approach to separate heterogeneously sized OMV subpopulations12,13,14. However, DGC is time-consuming, inefficient, and highly variable user-to-user22 and is, therefore, not ideal for scale-up. In contrast, size exclusion chromatography (SEC) represents a scalable, efficient, and consistent approach to purify OMVs21,23,24. We have found that a long (50-cm), gravity-flow, SEC column, filled with gel filtration medium is sufficient for efficiently purifying and separating subpopulations of OMVs. Specifically, we used this approach to separate A. actinomycetemcomitans OMVs into &#34;large&#34; and &#34;small&#34; subpopulations, as well as to remove protein and DNA contamination. Purification was completed in less than 4 h, and complete separation of the OMV subpopulations and removal of debris was accomplished.

<table><tbody><tr><td>1-Step Ultra TMB-ELISA</td><td>Thermo Scientific</td><td>34028</td><td></td></tr><tr><td>Amicon 50 kDa filters</td><td>Millipore Sigma</td><td>UFC905024</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Fisher Scientific</td><td>BP9704-100</td><td></td></tr><tr><td>ELISA Immuno Plates</td><td>Thermo Scientific</td><td>442404</td><td></td></tr><tr><td>FM 4-64</td><td>Thermo Scientific</td><td>T13320</td><td>1.5 x 50 cm</td></tr><tr><td>Glass Econo-Column</td><td>BioRad</td><td>7371552</td><td></td></tr><tr><td>Infinite 200 Pro Plate Reader</td><td>Tecan</td><td></td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Amresco (VWR)</td><td>0395-500G</td><td></td></tr><tr><td>Potassium Phosphate Monobasic Anhydrous (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Amresco (VWR)</td><td>0781-500G</td><td></td></tr><tr><td>Sephacryl S-1000 Superfine</td><td>GE Healthcare</td><td>17-0476-01</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Fisher Chemical</td><td>S271-3</td><td></td></tr><tr><td>Sodium Phosphate Dibasic Anhydrous (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Amresco (VWR)</td><td>0404-500G</td><td></td></tr><tr><td>Tris Base</td><td>VWR</td><td>0497-1KG</td><td></td></tr><tr><td>Tween&lt;sup&gt;(R)&lt;/sup&gt; 20</td><td>Acros Organics</td><td>23336-2500</td><td></td></tr></tbody></table>

size exclusion chromatography to analyze bacterial outer membrane vesicle heterogeneity

The cell wall of Gram-negative bacteria consists of an inner (cytoplasmic) and outer membrane (OM), separated by a thin peptidoglycan layer. Throughout growth, the outer membrane can bleb to form spherical outer membrane vesicles (OMVs). These OMVs are involved in numerous cellular functions including cargo delivery to host cells and communication with bacterial cells. Recently, the therapeutic potential of OMVs has begun to be explored, including their use as vaccines and drug delivery vehicles. Although OMVs are derived from the OM, it has long been appreciated that the lipid and protein cargo of the OMV differs, often significantly, from that of the OM. More recently, evidence that bacteria can release multiple types of OMVs has been discovered, and evidence exists that size can impact the mechanism of their uptake by host cells. However, studies in this area are limited by difficulties in efficiently separating the heterogeneously sized OMVs. Density gradient centrifugation (DGC) has traditionally been used for this purpose; however, this technique is time-consuming and difficult to scale-up. Size exclusion chromatography (SEC), on the other hand, is less cumbersome and lends itself to the necessary future scale-up for therapeutic use of OMVs. Here, we describe a SEC approach that enables reproducible separation of heterogeneously sized vesicles, using as a test case, OMVs produced by Aggregatibacter actinomycetemcomitans, which range in diameter from less than 150 nm to greater than 350 nm. We demonstrate separation of &#34;large&#34; (350 nm) OMVs and &#34;small&#34; (&#60;150 nm) OMVs, verified by dynamic light scattering (DLS). We recommend SEC-based techniques over DGC-based techniques for separation of heterogeneously sized vesicles due to its ease of use, reproducibility (including user-to-user), and possibility for scale-up.

Figure 2&#160;shows representative results from this method. OMVs produced by A. actinomycetemcomitans strain JP2 were first purified from the culture supernatant using ultracentrifugation15. We previously found that this strain produces two populations of OMVs, one with diameters of about 300 nm and one with diameters of about 100 nm15. To separate these OMV populations, we purified the sample using the SEC protocol described above. Each fraction was analyzed for lipid content using the lipophilic dye and for toxin (LtxA) content using enzyme-linked immunosorbent assay (ELISA) or an immunoblot. The lipid and toxin concentrations are reported as percentages, where &#34;%lipid&#34; indicates what percentage of the total lipid content of the sample is in each fraction and &#34;%toxin&#34; indicates what percentage of the total toxin content of the sample is in each fraction.
Figure 2A shows the averaged lipophilic dye results with standard deviations from three separate purifications, each performed by a different user, demonstrating the reproducibility of this technique. Two distinct lipid peaks are observed, corresponding to &#34;large&#34; OMVs (fraction number 13) and &#34;small&#34; OMVs (fraction number 25). We confirmed the size of the OMVs in these peaks using dynamic light scattering (DLS) and found the mean diameters of the OMVs in fractions 13 and 25 to be 296.6 nm and 142.6 nm, respectively, as shown in Fig. 2A. In comparison, the mean diameter of the OMV sample after ultracentrifugation but before SEC purification was previously found to be 161.0 nm15.
In Figure 2B, the amount of LtxA in each fraction, obtained using ELISA with a monoclonal antibody against LtxA25, is shown overlaid on the lipid concentration from panel A. This technique demonstrates that the toxin is associated primarily with one subpopulation of OMVs. Figure 2C shows the amount of LtxA in each fraction, measured using an immunoblot technique with the same anti-LtxA monoclonal antibody25, overlaid on the lipid concentration from panel A. While the overall trend is similar to what is observed in Figure 2B, the immunoblot approach is much less sensitive than the ELISA technique, resulting in noisier profiles. Figure 2D shows the percentage of the total protein concentration in each fraction, measured using the A280, overlaid on the lipid concentration profile. This panel demonstrates that SEC is able to remove significant amounts of free proteins from the OMV preparations, as evidenced by the high A280 values in fractions greater than 60. (In fact, most of the protein is found in these fractions, which do not contain OMVs, demonstrating that a large amount of protein co-purifies with the OMVs.) In addition, this result shows that the total protein concentration does not necessarily correlate with the concentration of specific proteins. In the case of A. actinomycetemcomtians OMVs, LtxA associates primarily with the population of larger OMVs, while more of the total protein associates with the smaller OMVs.
Together, these representative results demonstrate a number of important features of the SEC protocol for OMV purification. First, the technique is highly reproducible, even between users. Second, the use of a lipophilic dye to detect OMVs in each fraction is a simple and reliable method. Third, to detect specific protein concentrations, ELISA is more robust than an immunoblot. Fourth, SEC is able to remove large amounts of impurities, including proteins and nucleic acids.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62429/62429fig02.jpg" /> 
Figure 2: Representative results. A. actinomycetemcomitans JP2 OMVs were run through the SEC column and each fraction was analyzed for lipid content using a lipophilic dye and toxin (LtxA) content using a monoclonal antibody. (A) The average lipid content of each fraction reported as a percentage of the total lipid content, from three trials. Each data point represents the mean &#177; standard deviation. (B) The LtxA content of each fraction, reported as a percentage of the total LtxA content, as measured by ELISA with a monoclonal anti-LtxA antibody. (C) The LtxA content of each fraction, reported as a percentage of the total LtxA content, as measured by an immunoblot with a monoclonal anti-LtxA antibody. (D) The total protein content of each fraction, reported as a percentage of the total protein content, as measured by A280. Some of the data are reproduced from Chang et al.26 with permission from John Wiley and Sons Ltd. <a href="https://www.jove.com/files/ftp_upload/62429/62429fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity at JoVE.com

Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

Bacterial vesicles play important roles in pathogenesis and have promising biotechnological applications. The heterogeneity of vesicles complicates analysis and use; therefore, a simple, reproducible method to separate varying sizes of vesicles is necessary. Here, we demonstrate the use of size exclusion chromatography to separate heterogeneous vesicles produced by Aggregatibacter actinomycetemcomitans.

The cell wall of Gram-negative bacteria consists of an inner (cytoplasmic) and outer membrane (OM), separated by a thin peptidoglycan layer. Throughout ...

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Bioengineering

4.3K Views.  Lehigh University. Bacterial vesicles play important roles in pathogenesis and have promising biotechnological applications. The heterogeneity of vesicles complicates analysis and use; therefore, a simple, reproducible method to separate varying sizes of vesicles is necessary. Here, we demonstrate the use of size exclusion chromatography to separate heterogeneous vesicles produced by Aggregatibacter actinomycetemcomitans.

Video: Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

Biochemistry

Isolating Small Extracellular Vesicles and Extracting Their Peptidome

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

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

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

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

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

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.

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) ...

Immunology and Infection

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to assess the purity of the bilayers. The OM has an increased overall density compared to the inner membrane, largely due to the presence of lipooligosaccharides (LOS) and lipopolysaccharides (LPS) within the outer leaflet. LOS and LPS molecules are amphipathic glycolipids that have a similar structure, which consists of a lipid-A disaccharolipid and core-oligosaccharide substituent. However, only LPS molecules are decorated with a third subunit known as the O-polysaccharide, or O-antigen. The type and amount of glycolipids present will impact an organism&#8217;s OM density. Therefore, we tested whether the membranes of bacteria with varied glycolipid content could be similarly isolated using our technique. For the LPS-producing organisms, Salmonella enterica serovar Typhimurium and Escherichia coli, the membranes were easily isolated and the LPS O-antigen moiety did not impact bilayer partitioning. Acinetobacter baumannii produces LOS molecules, which have a similar mass to O-antigen deficient LPS molecules; however, the membranes of these microbes could not initially be separated. We reasoned that the OM of A. baumannii was less dense than that of Enterobacteriaceae, so the sucrose gradient was adjusted and the membranes were isolated. The technique can therefore be adapted and modified for use with other organisms.

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

Gram-negative bacteria produce two spatially segregated membranes. The outer membrane is partitioned from the inner membrane by a periplasm and a peptidoglycan layer. The ability to isolate the dual bilayers of these microbes has been critical for understanding their physiology and pathogenesis.

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to ...

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. ...

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain proteins, nucleic acids, and lipids that are reflective of the parent cell from which they are derived. Proper separation of EVs from other components in a sample allows for characterization of their associated cargo and lends insight into their potential as intercellular communicators and non-invasive biomarkers for numerous diseases. In the current study, oligodendrocyte derived EVs were isolated from cell culture media using a combination of state-of-the-art techniques, including ultrafiltration and size exclusion chromatography (SEC) to separate EVs from other extracellular proteins and protein complexes. Using commercially available SEC columns, EVs were separated from extracellular proteins released from human oligodendroglioma cells under both control and endoplasmic reticulum (ER) stress conditions. The canonical EV markers CD9, CD63, and CD81 were observed in fractions 1-4, but not in fractions 5-8. GM130, a protein of the Golgi apparatus, and calnexin, an integral protein of the ER, were used as negative EV markers, and were not observed in any fraction. Further, when pooling and concentrating fractions 1-4 as the EV fraction, and fractions 5-8 as the protein fraction, expression of CD63, CD81, and CD9 in the EV fraction was observed. The expression of GM130 or calnexin was not observed in either of the fraction types. The pooled fractions from both control and ER stress conditions were visualized with transmission electron microscopy and vesicles were observed in the EV fractions, but not in the protein fractions. Particles in the EV and protein fractions from both conditions were also quantified with nanoparticle tracking analysis. Together, these data demonstrate that SEC is an effective method for separating EVs from conditioned cell culture media.

The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography.

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Neuroscience

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 ...

Size Exclusion Chromatography Based Separation of Bacterial Extracellular Vesicles: A Technique to Separate Heterogenous Population of Gram-Negative Bacterial Outer Membrane Vesicles Based on Their Size

Source: Collins, S. M., et al., <a href="https://www.jove.com/v/62429">Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity</a>. J. Vis. Exp. (2021).
In this video, we demonstrate a technique&#8211;size exclusion chromatography&#8211;to separate a heterogenous population of outer membrane vesicles or OMVs derived from bacteria. These vesicles are nanosized spherical structures and are separated into large and small subpopulations based on their size through Size Exclusion Chromatography.

Source: Collins, S. M., et al., Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity. J. Vis. Exp. (2021).
In this ...

Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

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Chapters in this video

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

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

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

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

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

Size Exclusion Chromatography Based Separation of Bacterial Extracellular Vesicles: A Technique to Separate Heterogenous Population of Gram-Negative Bacterial Outer Membrane Vesicles Based on Their Size