eoe sub articles

EoE Sub Articles

1. Preparation of crude Mtb EV concentrate
NOTE: For detailed procedures on the cultivation of Mtb and preparation of culture filtrate protein (CFP), see References. It is recommended that bacterial culture media is free from growth supplements with EV-containing or proteinaceous components, such as Oleic Albumin Dextrose Catalase (OADC), and detergents such as Tween. It is also recommended that the bacterial culture&#39;s quality and harvested CFP be screened to ensure limited cell death and lysis.
<ol>
	<li>Prepare a 100 kDa molecular weight cut-off (MWCO) centrifugal filter (see Table of Materials) by adding the full volume capacity of phosphate-buffered saline (1x PBS). Centrifuge for 5 min at 2,800 x g at 4 &#176;C. 
	NOTE: If using a filter with no dead stop volume, care must be taken to ensure the sample volume does not reduce below the filter level, resulting in complete filter drying during centrifugation.</li>
	<li>Discard the flow-through and any remaining PBS before filling the sample chamber with Mtb CFP to its maximum capacity. Centrifuge at 2,800 x g at 4 &#176;C until the volume has reduced to the minimum volume of the ultrafiltration device. Repeat as necessary, adding more CFP to the unit until the entire sample has been reduced sufficiently. 
	NOTE: Retain a portion of the 100 kDa flow-through (100F) material for downstream qualification if desired. Store at 4 &#176;C.</li>
	<li>Add 1x PBS to the filter unit containing the concentrate up to the total device capacity. Reduce the volume as described in 1.2. Repeat this step five times to ensure complete washing and buffer exchange.</li>
	<li>Recover the 100 kDa concentrated CFP retentate (100R) according to the ultrafiltration device specifications (see Table of Materials). Once the 100R is recovered, wash the filter with a minimal volume of 1x PBS at least three times, and pool the wash with the 100R to maximize the recovery.</li>
	<li>Quantitate the 100R material with a bicinchoninic assay (BCA) following the manufacturer&#39;s instructions (see Table of Materials). To perform the assay, use several dilutions of samples 1:2, 1:5, and 1:10 in PBS. Test the sample in triplicate. 
	NOTE: Retain a portion of the 100R material for downstream qualification if desired. Store at 4 &#176;C.</li>
</ol>
2. Size exclusion chromatography for the enrichment of Mtb EVs from CFP
NOTE: The following procedure is specific for using 3 mg of 100R Mtb CFP with SEC column and automatic fraction collector (AFC, see Table of Materials). It can be adapted for other starting concentrations and column types by following the manufacturer&#39;s specifications. Additionally, the users are recommended to read and understand the automatic fraction collector user manual.
<ol>
	<li>Allow the SEC column to equilibrate to room temperature.</li>
	<li>Turn on the AFC using the power switch on the back of the tower unit and adjust the settings for the load cell, if necessary, by pressing SETUP on the main menu touchscreen. The load cell must only be calibrated upon first use, after every software update, and if inconsistency in fraction collection volumes is noticed.</li>
	<li>From the SETUP screen, align the carousel by selecting CAROUSEL &#62; CALIBRATE. Insert the carousel with the 13 small holes facing up into the AFC tower, and adjust the carousel so that the fluid nozzle is directly above the flush position by pressing the - and/or + buttons.</li>
	<li>Remove the caps from the equilibrated SEC column. Slide the SEC column into the appropriate column mount and carefully install it on the AFC tower. Ensure the radio frequency identification (RFID) tag on the column faces the AFC, and check that the connection between the column and the valve is secure. Place the waste outlet tubing in a collection container.</li>
	<li>From the SETUP screen, select Collection Schedule and set the count to 13 and the size to 0.5 mL by pressing the - and/or + buttons. Leave the &#34;Buffer Volume&#34; setting as the default of 2.7 mL, and then close the &#34;Collection Schedule&#34; window by pressing X.</li>
	<li>Select START COLLECTION from the home screen and confirm the collection parameters by selecting YES. Load 13 labeled 1.7 mL microcentrifuge tubes with the lids open and pointing toward the carousel center.</li>
	<li>Advance the AFC by selecting OK, then mount the column reservoir and advance again by pressing OK. Select the option to flush the column by pressing YES, and add one column volume of 0.2 &#181;m filtered PBS to remove any storage buffer. Once the flush is complete, advance the AFC by pressing OK.</li>
	<li>Place the carousel cover over the AFC and press OK. Prepare one column volume of 0.2 &#181;m filtered PBS. Use a pipette to remove any excess buffer from the column.</li>
	<li>Bring 3 mg of 100R sample as quantified in step 1.5 to 500 &#181;L with 1x PBS. Add the 3 mg sample to the top of the column. Advance the AFC and allow the sample to run into the frit. Once the sample has fully entered the column, add the PBS prepared in step 2.6 to the reservoir.</li>
	<li>Monitor the run of the AFC while it collects first the void volume and then the specified fractions. After the run completes and the carousel returns to waste position, remove the cover and remove the fraction tubes from the carousel. Store the fractions at 4 &#176;C prior to quantification (step 3) and qualification (step 4).</li>
	<li>If the column is to be reused, select the option to clean the column by pressing YES; otherwise, press NO. Follow the instructions on the AFC. 
	NOTE: Once the column is washed and the storage buffer has run through, it can be removed, capped, and stored at 4 &#176;C.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well plate</td>
			<td>Corning</td>
			<td>15705-066</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic Fraction Collector</td>
			<td>IZON Science</td>
			<td>AFC-V1-USD</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Allegra 6R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centricon Plus - 70 Centrifugal filter, 100 kDa cutoff</td>
			<td>Millipore Sigma</td>
			<td>UFC710008</td>
			<td>Ultrafiltration device used in step 1.1</td>
		</tr>
		<tr>
			<td>Phosphate-buffered Saline, 1X without calcium and magnesium</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>qEV Original 35 nm 5/pk</td>
			<td>IZON Science</td>
			<td>SP5-USD</td>
			<td>SEC column</td>
		</tr>
	</tbody>
</table>

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.

A Size Exclusion Chromatography Technique to Purify Mycobacterial Extracellular Vesicles

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

Take a size exclusion chromatography column packed with an inert matrix of polysaccharide resin particles. The porous particles have a defined pore size.
Flush the column with an equilibration buffer for optimal purification.
Add a concentrated culture filtrate processed from a culture of Mycobacterium tuberculosis &#160;&#8212; a pathogenic bacteria. The filtrate contains extracellular vesicles, or EVs, and other soluble proteins secreted by the bacteria.
EVs are heterogeneously sized, small, spherical structures. They contain macromolecules and virulence factors enclosed in a lipid bilayer.
The sample enters the matrix under gravity. Add the buffer to allow sample movement through the column.
The EVs cannot enter the resin due to the small pore size and instead move through the interparticle space, traveling a shorter path and eluting first.
Proteins, having a smaller size, enter the pores, travel a longer path through the resin, and elute at the end.
Collect the fractions containing EVs for downstream analysis.&#160;

a-size-exclusion-chromatography-technique-to-purify-mycobacterial

Universidad San Sebastian

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

In Vitro and In Vivo Models

115 Views. Source: Ryan, J. M., et al. Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography. 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 por...

Video: A Size Exclusion Chromatography Technique to Purify Mycobacterial Extracellular Vesicles - Concept

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

JoVE Journal

Biochemistry

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.

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 ...

Bioengineering

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.

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain ...

A Size Exclusion Chromatography Technique to Purify Mycobacterial Extracellular Vesicles

Transcript

A Size Exclusion Chromatography Technique to Purify Mycobacterial Extracellular Vesicles

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

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