This study was supported by funding from National Cancer Institute Grant CA-217833.

All studies involving animals were performed in accordance with a protocol that was approved by the Mayo Clinic Institutional Animal Care and Use Committee.
1. Bench Preparation
<ol>
	<li>Prepare a water bath and set it to 37 &#176;C. The other necessary equipment and set-up are shown in Figure 1.</li>
	<li>Measure 100 mg of collagenase type IV and add it to a 125 mL flask containing 100 mL of HBSS in a water bath (40 &#176;C). Ensure that the collagenase has been dissolved by swirling the liquid in the flask, and also make sure that the flask is submerged completely in the water bath. For greater efficiency, allow the collagenase to dissolve for at least 30 min even though it may appear to dissolve instantly.</li>
	<li>Submerge a 125 mL flask containing 50 mL of HBSS in a water bath at 40 &#176;C. This will be used for initial flushing.</li>
	<li>Spray down the surface of all instruments such as the scissors and forceps with 70% ethanol. Prepare a few clean cotton swabs.</li>
	<li>Rinse out the tubing of the pump by running 70% ethanol through the pump and remove any residual ethanol by rinsing it twice with running water.</li>
	<li>Lay an absorbent bench pad on the lab bench and place a hard box container on top. This will be needed to contain any excess liquids during the mouse perfusion. Wrap a polystyrene foam pad with aluminum foil and place it inside the container.</li>
	<li>Place a sterile 10 cm culture dish close to the bench. This will be used to hold the digested liver after the perfusion is completed.</li>
	<li>Pour 10 mL of collagenase medium (step 1.2) into a sterile culture dish in advance.</li>
	<li>Using a winged blood collection set, connect the 23-gauge to the free end of the pump tubing (cutting the wings off the butterfly cannula can lead to better handling). Pass until the tip of the cannula of blood collection is filled.</li>
</ol>
2. Animal Preparation
<ol>
	<li>Before starting anesthesia using isoflurane, make sure that an adequate amount of supply gas is available for the duration of the procedure. Turn on the oxygen (O2) supply to the induction chamber at 1-2 L, then turn on isoflurane between 2-4% using a flowmeter.</li>
	<li>Put the mouse into the induction chamber and close the top door. Monitor the mouse until it is recumbent. 
	NOTE: The gases in the chamber will keep the mice anesthetized for several minutes.</li>
	<li>Place the mouse on a polystyrene foam pad wrapped with aluminum foil. Switch the flow from the induction chamber to a nosecone. Ensure that anesthesia is adequate. If the mouse has started responding, gently restrain it in a nose cone until the it is fully anesthetized.</li>
	<li>Ensure anesthesia by monitoring respiration and response to stimulation during the procedure. Adjust the rate flowmeter as needed to ensure adequate anesthesia. The paws must be nonresponsive to the pinch test under anesthesia. Additional details may be obtained from the laboratory guide for the care of animals.</li>
	<li>Tape or pin down all four limbs of the mouse.</li>
	<li>Clean the skin over the abdomen by spraying with 70% ethanol and wiping it off with gauze and an alcohol pad. This step is critical to avoid contamination from the mouse fur.</li>
	<li>Make a wide-open cut through the skin from the anterior to the pelvis bone using a set of sterile scissors. Be careful not to cut any internal organs . Displace the intestines to the animal&#39;s left side&#160;by using a cotton tipped applicator to gently to expose the portal vein (PV) and inferior vena cava (IVC).</li>
</ol>
3. Cannulation and Perfusion (0.5 h)
<ol>
	<li>Using curved forceps, place a thread underneath the portal vein and tie a knot loosely to prepare cinching tightly after cannulation.</li>
	<li>Insert the cannula (23G from the blood collection set) into the portal vein 5-10 mm below the ligature. Do not insert the cannula past the first portal branch, otherwise the right anterior lobe can be inadequately perfused.The cannula can be fixed or fastened with thread using a stopper knot.</li>
	<li>Start the pump to infuse in HBSS at a low flow rate (1-2 mL/min). Once the cannulation is confirmed to be successful, the liver will begin to blanch.</li>
	<li>Cut the IVC to relieve pressure and allow excessive fluid within the liver to drain. This is best performed using the operators other hand so the cannula is not moved.</li>
	<li>Slowly increase the flow rate to 8 mL/min and complete the perfusion using the entire 50 mL volume of HBSS through the liver, over the next 5 min.</li>
	<li>Change the collagenase containing medium (step 1.2) into the beaker just before the HBSS is starting to run out. Ensure that air bubbles are not present and do not flow into the liver when changing the medium.</li>
	<li>Apply transient pressure to the IVC at 5 s intervals by clamping with forceps. This will cause the liver to swell and help with tissue digestion and dissociation (7-8 min). As digestion progresses, the liver will swell and become white. The liver can swell uniformly to approximately twice its original size.</li>
	<li>Turn off the pump and remove the cannula once digestion is completed. The completion of liver digestion will depend on the size of the mouse and condition of the liver. A dent in the liver can be observed if a cotton-tipped applicator is used to gently probe the liver.</li>
	<li>Remove the gallbladder from the liver, being careful not to tear it. Using a washed pair of scissors and forceps, extract the liver from the mouse into a sterile 10 cm culture dish containing phosphate-buffered saline (PBS) for surface washing. Transfer the liver carefully into the sterile 10 cm culture dish containing collagenase medium (from step 1.8). This is a critical step for avoiding contamination from the mouse blood and bile.</li>
	<li>Grab and tear apart the liver with two clean forceps while gently shaking the cells from the liver. As this happens, the medium will become clouded. All hepatocytes can be shaken away, leaving behind the connective tissue and vascular tissue.</li>
	<li>Triturate the cell solution many times using a 3 mL syringe until the undigested parts of the liver are shaken off. Pour it off into a 50 mL conical tube with 70 &#181;m nylon cell strainers to filter any undigested connective tissue. Wash the dish with HBSS to collect the remaining cells and fill up the 50 mL conical tube.</li>
	<li>Centrifuge softly the 50 mL conical tube in a swinging bucket rotor at 50 x g for 10&#160;min at 4 &#176;C.</li>
	<li>Transfer the supernatant to a new 50 mL conical tube.</li>
</ol>
4. Isolation of EVs (5 h)
<ol>
	<li>Centrifuge the supernatant at 300 x g for 10 min at 4 &#176;C. Transfer the supernatant into a new 50 mL conical tube.</li>
	<li>Centrifuge the supernatant at 2000 x g for 20 min at 4 &#176;C to remove cell debris and aggregates.</li>
	<li>Transfer the supernatant to a round-bottom tube and centrifuge the supernatant at 10,000 x g for 70 min at 4&#176;C.</li>
	<li>Collect the supernatant and place into a polycarbonate ultracentrifuge tube and centrifuge at 100,000 x g for 70 min at 4 &#176;C.</li>
	<li>Collect the pellet in an ultracentrifuge tube which is then washed by re-suspending in PBS. Centrifuge the supernatant further at 100,000 x g for 70 min at 4 &#176;C.</li>
	<li>The final pellet comprising of cellular nanovesicles can be directly used for experiments or re-suspended with 1000 &#181;L of PBS and stored at -80 &#176;C.</li>
</ol>
5. Assessment of Quality and Yield of Isolations
<ol>
	<li>Assess the size distribution and concentration using nanoparticle tracking analysis or tunable resistive pulse sensing following the instrument manufacturer&#39;s protocols.</li>
	<li>Perform further isolation and purification of specific vesicle populations by various approaches such as the addition of a sucrose gradient or cushion, immunoaffinity techniques, or size exclusion chromatography, based on specific experimental needs.</li>
</ol>

<ol>
	<li>Kogure, T., Lin, W. L., Yan, I. K., Braconi, C., Patel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+nanovesicle-mediated+microRNA+transfer:+a+mechanism+of+environmental+modulation+of+hepatocellular+cancer+cell+growth.">Intercellular nanovesicle-mediated microRNA transfer: a mechanism of environmental modulation of hepatocellular cancer cell growth.</a> Hepatology. 54 (4), 1237-1248 (2011).</li><li>Maji, S., Matsuda, A., Yan, I. K., Parasramka, M., Patel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+in+liver+diseases.">Extracellular vesicles in liver diseases.</a> American Journal of Physiology - Gastrointestinal and Liver Physiology. 312 (3), 194-200 (2017).</li><li>Kogure, T., Patel, T. <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+extracellular+nanovesicle+microRNA+from+liver+cancer+cells+in+culture.">Isolation of extracellular nanovesicle microRNA from liver cancer cells in culture.</a> Methods in Molecular Biology. 1024, 11-18 (2013).</li><li>Miller, L. L., Bly, C. G., Watson, M. L., Bale, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dominant+role+of+the+liver+in+plasma+protein+synthesis;+a+direct+study+of+the+isolated+perfused+rat+liver+with+the+aid+of+lysine-epsilon-C14.">The dominant role of the liver in plasma protein synthesis; a direct study of the isolated perfused rat liver with the aid of lysine-epsilon-C14.</a> Journal of Experimental Medicine. 94 (5), 431-453 (1951).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Methods in Cellular Biology. 13, 29-83 (1976).</li><li>Klaunig, J. E., 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=Mouse+liver+cell+culture.+I.+Hepatocyte+isolation.">Mouse liver cell culture. I. Hepatocyte isolation.</a> In Vitro. 17 (10), 913-925 (1981).</li><li>Li, W. C., Ralphs, K. L., Tosh, D. <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+culture+of+adult+mouse+hepatocytes.">Isolation and culture of adult mouse hepatocytes.</a> Methods in Molecular Biology. 633, 185-196 (2010).</li><li>Yin, Z., Ellis, E. C., Nowak, G. <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+mouse+hepatocytes+for+transplantation:+a+comparison+between+antegrade+and+retrograde+liver+perfusion.">Isolation of mouse hepatocytes for transplantation: a comparison between antegrade and retrograde liver perfusion.</a> Cell Transplantation. 16 (8), 859-865 (2007).</li><li>Thery, C., Amigorena, S., Raposo, G., Clayton, A. <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+characterization+of+exosomes+from+cell+culture+supernatants+and+biological+fluids.">Isolation and characterization of exosomes from cell culture supernatants and biological fluids.</a> Currents Protocols in Cell Biology. , (2006).</li><li>Raposo, G., 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=B+lymphocytes+secrete+antigen-presenting+vesicles.">B lymphocytes secrete antigen-presenting vesicles.</a> Journal of Experimental Medicine. 183 (3), 1161-1172 (1996).</li><li>Witwer, K. 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=Standardization+of+sample+collection,+isolation+and+analysis+methods+in+extracellular+vesicle+research.">Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.</a> Journal of Extracellular Vesicles. 2, (2013).</li></ol>

The authors have nothing to disclose.

This protocol describes an optimal and reproducible method for the isolation of hepatic tissue EV using a two-step perfusion process via the portal vein followed by differential ultracentrifugation. Important steps of the procedure include cannula placement, collagenase concentration and digestion time, flow speed of the medium, handling of the tissue after digestion, and classical differential ultracentrifugation.
Cell separation is achieved by separation from connective tissue components after digestion using collagenase type IV.The concentration of collagenase used for perfusion can range from 0.1 to 5 mg/mL. There can be considerable batch-to-batch variation in the efficacy of collagenase for tissue digestion. Concentrations of collagenase from 0.5 to 5 mg/mL were tested, but the concentration used did not have a major impact on the yield of EVs obtained. Using a higher concentration of collagenase will result in a more rapid swelling and whitening of the liver. The goal is to obtain satisfactory cell dissociation without excessive contamination or damage. An optimal concentration of collagenase used in these isolations is 1-2 mg/mL perfused for 7-8 min at a flow rate of 8 mL/min. A perfusion procedure that is too long will increase the risk of destroying the thin connective tissue within the liver as well as increase technical risks such as needle dislodgment from the portal vein or air trapping with the vein.
The most challenging aspect of this protocol is the cannulation of the portal vein. This can be challenging to perform, particularly in mice in the 18- to 25-g size range. The techniques for collagenase perfusion were originally developed for use in rats and subsequently adopted for use in mice after numerous modifications and adjustments. Cannulation using a 23G blood collection set is easier than placement of a catheter in blood vessels of small luminal diameter. Fixing the cannula using thread with a stopper knot is recommended to avoid dislodgement and the knot also serves as a marker of portal vein location in case the cannula comes off the vessel.
For downstream analysis, it is extremely important to have minimal contamination from cells. There are several important considerations in the handling of tissue after digestion. First, the forceps and scissors are changed when the liver is extracted to avoid blood contamination. Second, it is critical that the gallbladder be carefully removed from the liver to avoid tearing and unwanted contamination from bile. Third, once the liver has been removed from the mouse, the liver is washed very gently using PBS to remove any blood. Minimizing contamination with cells should be given a higher priority than reduction in the yield of EVs obtained.
Ultracentrifugation is the most commonly used method for the isolation and purification of EVs8,9,10,11. This approach will remove most parenchymal cells such as hepatocytes or cholangiocytes and non-parenchymal cells such as Kupffer cells, sinusoidal endothelial cells, and stellate cells, In addition, cell debris, cell aggregations, and dead cells will also be removed by differential centrifugation. Further purification and isolation of specific populations can be performed by size exclusion chromatography to remove any non-vesicular protein aggregates or lipoproteins.
A limitation of this protocol is that it may not capture all tissue vesicles, given the possibility that some vesicles may be removed in the perfusate. If a global assessment is needed, collection of perfusate and isolation of vesicles within perfusate should be considered. A further limitation is the potential for cell damage. To monitor the potential impact of excessive cell death, cell viability can be monitored and incorporated within quality parameters for tissue EV isolations. In conclusion, this procedure describes an optimized workflow using a two-step perfusion technique via the portal vein followed by differential ultracentrifugation for obtaining liver tissue EVs from mouse livers at a high yield. These tissue EVs are suitable for downstream analyses such as characterization of biomolecular composition and other studies that aim to characterize their physiological or pathophysiological roles or potential applications as disease markers.

Extracellular vesicles (EVs) are membrane-bound vesicles that are released from many different types of cells in the body. EVs contain a cargo of molecules that include RNA, DNA, and protein. Transfer of this cargo by EVs from one cell to another is postulated as one mechanism by which cells within tissues communicate with each other1. The majority of information regarding the cargo or roles of EVs in normal health and diseases has been derived from studies on EVs obtained from cells in culture or collected from the circulation or other body fluids2. In order to understand their physiological roles in vivo, a robust method is necessary for the isolation of tissue EVs that captures all populations of EVs and avoids cellular damage or contamination3. The overall goal of the method described herein is to isolate tissue EVs from mouse livers.
Most cell types in the liver have been shown to produce EVs, and the study of EV-based signaling is advancing basic knowledge and understanding of hepatic diseases. However, the combined impact of EVs from different cell types within tissues is only partially understood. Isolation of EVs from liver tissues is necessary in order to understand the in situ contributions of EVs within the tissue milieu. The approach described herein is based on a two-step perfusion to enhance tissue dissociation and minimize cell damage. Subsequently, EVs are isolated from the dissociated liver tissue. Approaches using two-step perfusion for isolation of hepatocytes have been used since the early 1950s4. These methods for hepatocyte isolation have been modified and continuously improved and are now standard approaches for isolation of hepatocytes in cultures, in cell suspensions, and from tissues5,6,7. In the first step, the liver is subjected to a non-recirculating perfusion with calcium-free buffer,Hank&#39;s balanced salt solution (HBSS). In the second step, the liver is perfused with collagenase to dissolve the extracellular matrix for further separation of desmosomal cell-to-cell junctions. An optimal treatment time for collagenase dissolution is 7 to 10 minutes. A shorter duration of treatment will cause incomplete dissolution and retain cell contacts in the liver, whereas a longer duration may cause liver damage or portal vein disruption. EVs are then isolated using differential centrifugation to remove cells and cellular debris. This results in EV collection in high yields that can be used for further downstream analyses or studies.

<table border="0" cellpadding="0" cellspacing="0" style="width:783px;" width="783">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:432px;">125 mL Erlenmeyer flask</td>
			<td style="width:168px;">Fisher scientific</td>
			<td style="width:119px;">FB500125</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">125 mL Erlenmeyer flask</td>
			<td>Fisher scientific</td>
			<td>FB500125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:432px;">Curved non-serrated scissors</td>
			<td style="width:168px;">Fine Science Tools</td>
			<td style="width:119px;">14069-12</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:432px;">Curved forceps</td>
			<td style="width:168px;">Fine Science Tools</td>
			<td style="width:119px;">13009-12</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:432px;">Masking tape</td>
			<td style="width:168px;">Home supply store</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:432px;">Aluminum foil</td>
			<td style="width:168px;">Home supply store</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:432px;">Styrofoam pad</td>
			<td style="width:168px;">Home supply store</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Absorbent Bench Underpad</td>
			<td style="width:168px;">Scientific inc.</td>
			<td>B1623</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:432px;">Masterflex L/S Digital Miniflex Pump</td>
			<td style="width:168px;">Cole-parmer</td>
			<td>ZX-07525-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:432px;">Water bath</td>
			<td>Thermo Electron Precision</td>
			<td>2837</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:432px;">Blood collection sets</td>
			<td style="width:168px;">Becton Dickinson</td>
			<td style="width:119px;">367292</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:432px;">Petri dish, clear lid 100x15</td>
			<td style="width:168px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875712</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Falcon 70mm Nylon Cell Strainers</td>
			<td>Fisher scientific</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:432px;">50 mL conical tubes</td>
			<td style="width:168px;">Fisher Scientific</td>
			<td style="width:119px;">12-565-270</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cotton Tipped Applicators</td>
			<td style="width:168px;">Moore medical</td>
			<td>69622</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:432px;">25mL Serological Pipet</td>
			<td style="width:168px;">Falcon</td>
			<td style="width:119px;">357525</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ohmeda Isotec 4 Isoflurane Vaporiser</td>
			<td>BioSurplus</td>
			<td>203-2751</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:432px;">O2 gas</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:432px;">Isoflurane</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:432px;">&#160;Levo Plus Motorized Pipette Filler</td>
			<td>Scilogex</td>
			<td>74020002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge 5804R</td>
			<td style="width:168px;">Sigma-aldrich</td>
			<td>22628048</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Beckman Coulter Optima L-100 XP</td>
			<td>Beckman</td>
			<td>969347</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Beckman Coulter Avanti JXN-26</td>
			<td>Beckman</td>
			<td>B34182</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">70 Ti Fixed-Angle Rotor</td>
			<td>Beckman</td>
			<td>337922</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">JA-25.50Fixed-Angle Rotor</td>
			<td>Beckman</td>
			<td>363055</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nalgene Round-bottom tube</td>
			<td>Thermo Scientific</td>
			<td>3118-0028</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polycarbonate ultracentrifuge tubes with cap assembly</td>
			<td>Beckman</td>
			<td>355618</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:432px;">Reagents</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">HyClone Hank&#39;s Balanced Salt Solution (HBSS), Ca/Mg free</td>
			<td>Fisher scientific</td>
			<td>SH30588.01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase, Type IV, powder</td>
			<td>Fisher scientific</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline (PBS)</td>
			<td>Fisher scientific</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of tissue extracellular vesicles from the liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

The apparatus needed for these isolations comprises of standard laboratory equipment, making this a relatively simple and cost-effective approach. Isolations have been performed from twelve- to thirty-week-old male and female Balb/c or FVB mice. The tray holding the mouse is lined with aluminum foil inside a hard-walled container that collects excess fluids during the perfusion. The flasks containing HBSS or collagenase-containing medium are submerged in a water bath (40 &#176;C) ready to be used. Two sterile 10 cm culture dishes are used. One is needed for surface washing with PBS, and the other for hepatocyte separation from the connective tissue components.
In this method, the liver is perfused in a non-continuous manner via the portal vein in preference to cannulation from the inferior vena cava. An alternative and commonly used perfusion approach is to perform retrograde perfusion by cannulating the inferior vena cava and cutting the portal vein for drainage. However, portal vein cannulation is easy to access and involves a short distance to the liver, as the portal vein feeds directly into the liver8. The selection of an insertion point for cannulation is crucial for optimal success (Figure 2). The cannula is placed past the branches of the stomach and pancreatic veins but not beyond the first portal branch (right and left hepatic portal veins).Once the optimal insertion location in the portal vein is identified, curved forceps are used to place a thread underneath portal vein and tie a loose knot. The needle of cannula is fixed with thread using a stopper knot to stop the needle from falling out.
Figure 3&#160;outlines the overall processing scheme for the differential centrifugation for liver tissue EVs isolation. Ultracentrifugation removes cells, debris and other impurities. The first four centrifugation steps (50 x g, 300 x g, 2,000 x g, 10,000 x g) are designed to remove hepatocytes, intact other cells, dead cells, or cell debris respectively. (Figures 4A and 4B). After these steps, ultracentrifugation is again performed at 100,000 x g to collect the pellet (Figure 4C). The pellet is washed by re-suspending in PBS and subjected to a final ultracentrifugation at 100,000 x g. The pellet after ultracentrifugation is absolutely visible and viscid in this procedure compared to EVs from conditioned culture media. Pipetting many times is required until any brown aggregates are out of sight and completely dissolved. The final pellet is re-suspended with 1000 &#181;L of PBS (Figure 4D). Ultracentrifugation removes impurities and other soluble contaminants from the plasma, which can affect functional experimental outcomes. The centrifugation is be carried out at 4 &#730;C.
From mouse liver, this method yields a tissue EV concentration that ranges from 1.74 to 4.00 x 1012 with a mean of 3.46 x 1012 particles per mL as determined by nanoparticle tracking analysis (NTA) (Figure 5). The mean size of the isolated liver tissue EVs was 157.7 nm, with a mode size of 144.5 nm and EV sizes ranging from 100-600 nm by NTA. The yield of EV will depend on factors such as the liver weight and losses within the perfusate or ultracentrifugation steps.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58649/58649fig1.jpg" /> 
Figure 1: Bench preparation. The materials and their locations are: (A) pump, (B) warmed 125 mL flask containing HBSS and water suction port, (C) nose cone connected to an Isoflurane vaporiser, (D) water exhaust port of the pump connecting with needle, (E) tray lined with aluminum foil inside a hard walled container, and (F) 10 cm culture dish in which collagenase medium is poured in advance. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58649/58649fig1large.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/58649/58649fig2.jpg" /> 
Figure 2: Cannulation site. The anatomy of the mouse abdomen is shown. Using curved forceps, a thread is placed underneath the portal vein (PV) and a loose knot is tied. The insertion location is near the liver, 5-10 mm below the ligature, but not beyond the first portal branch (left and right hepatic portal veins). The cannula is fixed or fastened using thread with a stopper knot. This knot serves as a marker of the PV location if the cannula is dislodged. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58649/58649fig2large.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/58649/58649fig3.jpg" /> 
Figure 3: Schematic of centrifugation steps. The goal is to remove unwanted cells and other components and isolate EVs. The first four centrifugation steps are designed to remove hepatocytes and other cells, dead cells, or cell debris using differential centrifugation. After these steps, ultracentrifugation is performed at 100,000 x g to collect the pellet of EVs. The pellet is washed by re-suspending in PBS and subjected to a final ultracentrifugation at 100,000 x g. All the centrifugation steps are carried out at 4 &#730;C. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58649/58649fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58649/58649fig4.jpg" /> 
Figure 4: Differential centrifugation. (A) After centrifugation at 50 x g for 10 min, a pellet containing hepatocytes is observed. (B) A round-bottom tube is used for centrifugation at 10,000 x g for 70 min to remove cell debris. (C) A polycarbonate ultracentrifuge tube is used for centrifugation at 100,000 x g for 70 min. The pellet is collected in one tube and washed by re-suspending with PBS. (D) The final pellet is re-suspended in 1000 &#181;L of PBS. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58649/58649fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58649/58649fig5.jpg" /> 
Figure 5: Representative result. The size and concentration of liver tissue EVs can be determined by nanoparticle tracking analysis (NTA). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58649/58649fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation of Tissue Extracellular Vesicles from the Liver at JoVE.com

Isolation of Tissue Extracellular Vesicles from the Liver

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

isolation-of-tissue-extracellular-vesicles-from-the-liver

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.4K Views.  Mayo Clinic. This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

Video: Isolation of Tissue Extracellular Vesicles from the Liver

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to produce adenosine triphosphate (ATP). The isolation of the intact F1-ATPase from its native source is an essential prerequisite to characterize the enzyme&#39;s protein composition, kinetic parameters, and sensitivity to inhibitors. A highly pure and homogeneous F1-ATPase can be used for structural studies, which provide insight into molecular mechanisms of ATP synthesis and hydrolysis. This article describes a procedure for the purification of the F1-ATPase from Trypanosoma brucei, the causative agent of African trypanosomiases. The F1-ATPase is isolated from mitochondrial vesicles, which are obtained by hypotonic lysis from in vitro cultured trypanosomes. The vesicles are mechanically fragmented by sonication and the F1-ATPase is released from the inner mitochondrial membrane by the chloroform extraction. The enzymatic complex is further purified by consecutive anion exchange and size-exclusion chromatography. Sensitive mass spectrometry techniques showed that the purified complex is devoid of virtually any protein contaminants and, therefore, represents suitable material for structure determination by X-ray crystallography or cryo-electron microscopy. The isolated F1-ATPase exhibits ATP hydrolytic activity, which can be inhibited fully by sodium azide, a potent inhibitor of F-type ATP synthases. The purified complex remains stable and active for at least three days at room temperature. Precipitation by ammonium sulfate is used for long-term storage. Similar procedures have been used for the purification of F1-ATPases from mammalian and plant tissues, yeasts, or bacteria. Thus, the presented protocol can serve as a guideline for the F1-ATPase isolation from other organisms.

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

This protocol describes the purification of F1-ATPase from the cultured insect stage of Trypanosoma brucei. The procedure yields a highly pure, homogeneous, and active complex suitable for structural and enzymatic studies.

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to ...

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released from many cell types and play a pivotal role in regulating cell-cell communication and a diverse range of biological processes. Many different methods for the characterization of EVs have been described. However, most of these methods have the disadvantage that the preparation and characterization of the samples are very time-consuming, or it is extremely difficult to analyze specific markers of interest due to their small size and due to the lack of discrete populations. While methods for analysis of EVs have been considerably improved over the last decade, there is still no standardized method for characterization of single EVs. Here, we demonstrate a semi-automated method for characterization of single EVs by fluorescence-based nanoparticle-tracking analysis. The protocol that is presented addresses the common problem of many researchers in this field and provides the complete workflow for rapid isolation of EVs and characterization with PKH67, a general cell membrane linker, as well as with specific surface markers such as CD63, CD9, vimentin, and lysosomal-associated membrane protein 1 (LAMP-1). The presented results show a high level of reproducibility, as confirmed by other methods, such as Western blotting. In the conducted experiments, we exclusively used EVs isolated from human serum samples, but this method is also suitable for plasma or other body fluids and can be adjusted for characterization of EVs from cell culture supernatants. Irrespective of the future progress of research on EV biology, the protocol that is presented here provides a rapid and reliable method for rapid characterization of single EVs with specific markers.

In this protocol, we describe the complete workflow for rapid isolation of extracellular vesicles from human whole blood and characterization of specific markers by fluorescence-based nanoparticle-tracking analysis. The presented results show a high level of reproducibility and can be adjusted to cell culture supernatants.

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in liver cancer, the modification by SUMO (Small Ubiquitin MOdifier) has been given the most attention. Isolation of endogenous SUMOylated proteins in vivo is challenging due to the presence of active SUMO-specific proteases. Initial studies of SUMOylation in vivo were based on the molecular detection of specific SUMOylated proteins (e.g., by western blot). However, in many cases, antibodies, generally made with non-modified recombinant protein, did not immunoprecipitate SUMOylated forms of the protein of interest.&#160;Nickel chromatography has been the other approach to study SUMOylation by capturing histidine-tagged versions of SUMO molecules. This approach is mainly used in cells stably expressing or transiently transfected with His-SUMO molecules. To overcome these limitations, SUMO-binding entities (SUBEs) were developed to isolate endogenous SUMOylated proteins. Herein, we describe all the steps required for the enrichment, isolation, and identification of SUMOylated substrates from human hepatoma cells and hepatic tissues from a liver cancer mouse model by using SUBEs. Firstly, we describe methods involved in the preparation and lysis of the human hepatoma cells and liver tumor tissue samples. Then, a thorough explanation of the preparation of SUBEs and controls is detailed along with the protocol for the protein pull-down assays. Finally, some examples are provided regarding the options available for the identification and characterization of the SUMOylated proteome, namely the use of western-blot analysis for the detection of a specific SUMOylated substrate from liver tumors or the use of proteomics by mass spectrometry for high-throughput characterization of the SUMOylated proteome and interactome in hepatoma cells.

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Here, we present a protocol to enrich, isolate, identify, and characterize proteins modified by SUMO in vivo both from human hepatoma cells and liver tumors obtained from mouse models of hepatocellular carcinoma by using SUMO-binding entities (SUBEs).

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. ...

Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. Biomolecules such as proteins, carbohydrates, vitamins, fatty acids, nucleic acids, and enzymes are fundamental building blocks of living beings; they assemble into complex networks in biosystems to synchronize a myriad of life events. Proteins typically utilize complex interactome networks to carry out their functions; hence it is mandatory to evaluate such interactions to unravel their importance in cells at both cellular and organism levels. Toward this goal, we introduce a rapidly emerging technology, field-effect biosensing (FEB), to determine specific biomolecular interactions. FEB is a benchtop, label-free, and reliable biomolecular detection technique to determine specific interactions and uses high-quality electronic-based biosensors. The FEB technology can monitor interactions in the nanomolar range due to the biocompatible nanomaterials used on its biosensor surface. As a proof of concept, the protein-protein interaction (PPI) between heat shock protein 90 (Hsp90) and cell division cycle 37 (Cdc37) was elucidated. Hsp90 is an ATP-dependent molecular chaperone that plays an essential role in the folding, stability, maturation, and quality control of many proteins, thereby regulating multiple vital cellular functions. Cdc37 is regarded as a protein kinase-specific molecular chaperone, as it specifically recognizes and recruits protein kinases to Hsp90 to regulate their downstream signal transduction pathways. As such, Cdc37 is considered a co-chaperone of Hsp90. The chaperone-kinase pathway (Hsp90/Cdc37 complex) is hyper-activated in multiple malignancies promoting cellular growth; therefore, it is a potential target for cancer therapy. The present study demonstrates the efficiency of FEB technology using the Hsp90/Cdc37 model system. FEB detected a strong PPI between the two proteins (KD values of 0.014 &#181;M, 0.053 &#181;M, and 0.072 &#181;M in three independent experiments). In summary, FEB is a label-free and cost-effective PPI detection platform, which offers fast and accurate measurements.

Field-effect biosensing (FEB) is a label-free technique for detecting biomolecular interactions. It measures the electric current through the graphene biosensor to which the binding targets are immobilized. The FEB technology was used to evaluate biomolecular interactions between Hsp90 and Cdc37 and a strong interaction between the two proteins was detected.

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. ...

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 &#181;m nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.

This protocol provides a rapid and size-specific isolation method for small extracellular vesicles by optimizing the size of the air spray nozzle, sheath fluid pressure, sample flow pressure, voltage, gain, and triggering threshold parameters.

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...