Name: size exclusion chromatography to analyze bacterial outer membrane vesicle heterogeneity
Uploaded: 2021-03-31T15:00:00
Description: Watch this Scientific Journal Video about Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity at JoVE.com

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 Na2HP...

<ol>
	<li>Kuehn, M. J., Kesty, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+outer+membrane+vesicles+and+the+host-pathogen+interaction.">Bacterial outer membrane vesicles and the host-pathogen interaction.</a> Genes and Development. 19, 2645-2655 (2005).</li><li>Kulp, A., Kuehn, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+functions+and+biogenesis+of+secreted+bacterial+outer+membrane+vesicles.">Biological functions and biogenesis of secreted bacterial outer membrane vesicles.</a> Annual Reviews Microbiology. 64, 163-184 (2010).</li><li>Kato, S., Kowashi, Y., Demuth, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+membrane-like+vesicles+secreted+by+Actinobacillus+actinomycetemcomitans+are+enriched+in+leukotoxin.">Outer membrane-like vesicles secreted by Actinobacillus actinomycetemcomitans are enriched in leukotoxin.</a> Microbial Pathogenesis. 32 (1), 1-13 (2002).</li><li>Nice, J. B., 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=Aggregatibacter+actinomycetemcomitans+leukotoxin+is+delivered+to+host+cells+in+an+LFA-1-independent+manner+when+associated+with+outer+membrane+vesicles.">Aggregatibacter actinomycetemcomitans leukotoxin is delivered to host cells in an LFA-1-independent manner when associated with outer membrane vesicles.</a> Toxins. 10 (10), 414 (2018).</li><li>Haurat, M. F., 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=Selective+sorting+of+cargo+proteins+into+bacterial+membrane+vesicles.">Selective sorting of cargo proteins into bacterial membrane vesicles.</a> Journal of Biological Chemistry. 286 (2), 1269-1276 (2011).</li><li>Horstman, A. L., Kuehn, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enterotoxigenic+Escherichia+coli+secretes+active+heat-labile+enterotoxin+via+outer+membrane+vesicles.">Enterotoxigenic Escherichia coli secretes active heat-labile enterotoxin via outer membrane vesicles.</a> The Journal of Biological Chemistry. 275 (17), 12489-12496 (2000).</li><li>Wai, S. N., 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=Vesicle-mediated+export+and+assembly+of+pore-forming+oligomers+of+the+Enterobacterial+ClyA+cytotoxin.">Vesicle-mediated export and assembly of pore-forming oligomers of the Enterobacterial ClyA cytotoxin.</a> Cell. 115, 25-35 (2003).</li><li>Balsalobre, C., 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=Release+of+the+Type+I+secreted+&#945;-haemolysin+via+outer+membrane+vesicles+from+Escherichia+coli.">Release of the Type I secreted &#945;-haemolysin via outer membrane vesicles from Escherichia coli.</a> Molecular Microbiology. 59 (1), 99-112 (2006).</li><li>Donato, G. M., 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=Delivery+of+Bordetella+pertussis+adenylate+cyclase+toxin+to+target+cells+via+outer+membrane+vesicles.">Delivery of Bordetella pertussis adenylate cyclase toxin to target cells via outer membrane vesicles.</a> FEBS Letters. 586, 459-465 (2012).</li><li>Kim, Y. 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=Outer+membrane+vesicles+of+Vibrio+vulnificus+deliver+cytolysin-hemolysin+VvhA+into+epithelial+cells+to+induce+cytotoxicity.">Outer membrane vesicles of Vibrio vulnificus deliver cytolysin-hemolysin VvhA into epithelial cells to induce cytotoxicity.</a> Biochemical and Biophysical Research Communications. 399, 607-612 (2010).</li><li>Maldonado, R., Wei, R., Kachlany, S. C., Kazi, M., Balashova, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+effects+of+Kingella+kingae+outer+membrane+vesicles+on+human+cells.">Cytotoxic effects of Kingella kingae outer membrane vesicles on human cells.</a> Microbial Pathogenesis. 51 (1-2), 22-30 (2011).</li><li>Turner, L., 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=Helicobacter+pylori+outer+membrane+vesicle+size+determines+their+mechanisms+of+host+cell+entry+and+protein+content.">Helicobacter pylori outer membrane vesicle size determines their mechanisms of host cell entry and protein content.</a> Frontiers in Immunology. 9, 1466 (2018).</li><li>Zavan, L., Bitto, N. J., Johnston, E. L., Greening, D. W., Kaparakis-Liaskos, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+growth+stage+determines+the+size,+protein+composition,+and+preferential+cargo+packaging+of+outer+membrane+vesicles.">Helicobacter pylori growth stage determines the size, protein composition, and preferential cargo packaging of outer membrane vesicles.</a> Proteomics. 19 (1-2), 1800209 (2019).</li><li>Rompikuntal, P. K., 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=Perinuclear+localization+of+internalized+outer+membrane+vesicles+carrying+active+cytolethal+distending+toxin+from+Aggregatibacter+actinomycetemcomitans.">Perinuclear localization of internalized outer membrane vesicles carrying active cytolethal distending toxin from Aggregatibacter actinomycetemcomitans.</a> Infections and Immunity. 80 (1), 31-42 (2012).</li><li>Nice, J. B., 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=Aggregatibacter+actinomycetemcomitans+leukotoxin+is+delivered+to+host+cells+in+an+LFA-1-independent+manner+when+associated+with+outer+membrane+vesicles.">Aggregatibacter actinomycetemcomitans leukotoxin is delivered to host cells in an LFA-1-independent manner when associated with outer membrane vesicles.</a> Toxins. 10 (10), 414 (2018).</li><li>Stevenson, T. C., 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=Immunization+with+outer+membrane+vesicles+displaying+conserved+surface+polysaccharide+antigen+elicits+broadly+antimicrobial+antibodies.">Immunization with outer membrane vesicles displaying conserved surface polysaccharide antigen elicits broadly antimicrobial antibodies.</a> Proceedings of the National Academy of Sciences. 115 (14), 3106-3115 (2018).</li><li>Gujrati, V., 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=Bioengineered+bacterial+outer+membrane+vesicles+as+cell-specific+drug-delivery+vehicles+for+cancer+therapy.">Bioengineered bacterial outer membrane vesicles as cell-specific drug-delivery vehicles for cancer therapy.</a> ACS Nano. 8 (2), 1525-1537 (2014).</li><li>Huang, W., 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=Development+of+novel+nanoantibiotics+using+an+outer+membrane+vesicle-based+drug+efflux+mechanism.">Development of novel nanoantibiotics using an outer membrane vesicle-based drug efflux mechanism.</a> Journal of Controlled Release. 317, 1-22 (2020).</li><li>Chen, D. 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=Delivery+of+foreign+antigens+by+engineered+outer+membrane+vesicle+vaccines.">Delivery of foreign antigens by engineered outer membrane vesicle vaccines.</a> Proceedings of the National Academy of Sciences. 107 (7), 3099-3104 (2010).</li><li>Chen, L., 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=Outer+membrane+vesicles+displaying+engineered+glycotopes+elicit+protective+antibodies.">Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies.</a> Proceedings of the National Academy of Sciences. 113 (26), 3609-3618 (2016).</li><li>Singorenko, P. D., 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 (1), 1324731 (2017).</li><li>Zeringer, E., Barta, T., Li, M., Vlassov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+isolation+of+exosomes.">Strategies for isolation of exosomes.</a> Cold Spring Harbor Protocols. 2015 (4), 319-323 (2015).</li><li>Benedikter, B. 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=Ultrafiltration+combined+with+size+exclusion+chromatography+efficiently+isolates+extracellular+vesicles+from+cell+culture+media+for+compositional+and+functional+studies.">Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies.</a> Science Reports. 7 (1), 15297 (2017).</li><li>Mol, E. A., Goumans, M. J., Doevendans, P. A., Sluijter, J. P. G., Vader, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Higher+functionality+of+extracellular+vesicles+isolated+using+size-exclusion+chromatography+compared+to+ultracentrifugation.">Higher functionality of extracellular vesicles isolated using size-exclusion chromatography compared to ultracentrifugation.</a> Nanomedicine. 13 (6), 2061-2065 (2017).</li><li>Lally, E. T., Golub, E. E., Kieba, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+immunological+characterization+of+the+domain+of+Actinobacillus+actinomycetemcomitans+leukotoxin+that+determines+its+specificity+for+human+target+cells.">Identification and immunological characterization of the domain of Actinobacillus actinomycetemcomitans leukotoxin that determines its specificity for human target cells.</a> Journal of Biological Chemistry. 269 (49), 31289-31295 (1994).</li><li>Chang, E. H., Giaquinto, P., Huang, J., Balashova, N. V., Brown, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigallocatechin+gallate+inhibits+leukotoxin+release+by+Aggregatibacter+actinomycetemcomitans+by+promoting+association+with+the+bacterial+membrane.">Epigallocatechin gallate inhibits leukotoxin release by Aggregatibacter actinomycetemcomitans by promoting association with the bacterial membrane.</a> Molecular Oral Microbiology. 35 (1), 29-39 (2020).</li><li>Klimentov&#225;, J., Stul&#237;k, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+isolation+and+purification+of+outer+membrane+vesicles+from+gram-negative+bacteria.">Methods of isolation and purification of outer membrane vesicles from gram-negative bacteria.</a> Microbiological Research. 170, 1-9 (2015).</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 (1), 1324731 (2017).</li><li>Mongui&#243;-Tortajada, M., G&#225;lvez-Mont&#243;n, C., Bayes-Genis, A., Roura, S., Borr&#224;s, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicle+isolation+methods:+rising+impact+of+size-exclusion+chromatography.">Extracellular vesicle isolation methods: rising impact of size-exclusion chromatography.</a> Cellular and Molecular Life Sciences. 76 (12), 2369-2382 (2019).</li></ol>

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 equilib...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 (KH2PO4)</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 (Na2HPO4)</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(R) 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. E...

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 ...

Research

JoVE Journal

Bioengineering

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Bacterial Detection & Identification Using Electrochemical Sensors

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different ...