This study was supported by a K08 Award (DK090154-01) from the National Institutes of Health (NIH; to F.M.S.).

Obtaining the bile from patients by endoscopic retrograde cholangiopancreatography (ERCP) requires the approval of a human subjects study protocol by the Institutional Review Board. All work presented here was approved by the Johns Hopkins University Institutional Review Board.
1. Exosome Isolation from Bile
<ol>
	<li>Perform endoscopic retrograde cholangiopancreatography (ERCP). Place the patient in the supine position and intubate.
	<ol>
		<li>Pass a duodenoscope through the mouth of the patient and insert it into the second part of the duodenum and identify the major papilla.</li>
		<li>Selectively cannulate the common bile duct by inserting a triple lumen sphincterotome pre-loaded with a 0.035 inch (0.9 mm) hydrophilic guidewire.</li>
		<li>Advance the guidewire under fluoroscopic guidance to the left hepatic duct since on occasion it can be difficult to differentiate the cystic duct from the right hepatic duct, then advance the sphincterotome 5 cm into the bile duct to acquire a stable position.</li>
		<li>Remove the guidewire, attach a 10 ml syringe to the guidewire port through the Luer lock and aspirate the bile using 10 ml of negative pressure.</li>
		<li>Remove the syringe containing the bile to proceed with the ERCP by reinserting the guidewire and injecting contrast agent through the injection port to opacify the biliary tree.</li>
		<li>Transfer the collected bile into a 15 ml tube on ice and keep on ice until ready to proceed with the centrifugation step. 
		Caution: Wear gloves to protect yourself and the samples! Treat the bile as a potential source of infection as it can contain Hepatitis A viral particles!</li>
		<li>Keep the sample(s) on ice. Either proceed with step 1.2 or centrifuge at 500 x g at 4 &#176;C for 10 min and then freeze at -80 &#176;C until further use. 
		Note: Samples stored this way for several years have routinely been used with good results19.</li>
	</ol>
	</li>
	<li>Transfer 400 &#181;l of bile into a microcentrifuge tube and collect cells and cell debris by centrifugation at 300 x g for 10 min at 4 &#176;C.</li>
	<li>Remove the supernatant by pipetting, transfer to a fresh microcentrifuge tube and centrifuge the sample(s) at 16,500 x g for another 20 min at 4 &#176;C to clear the supernatant of further debris. Discard the tubes in the biohazard waste. Alternatively, spin the samples in an ultracentrifuge instead.</li>
	<li>Collect the supernatant and filter it in a biosafety cabinet with a syringe through a 0.2 &#181;m polyethersulfone (PES) membrane filter which has good flow rates and low protein binding to remove any particles larger than 200 nm left.</li>
	<li>Pipet the filtered supernatant to ultracentrifuge tubes and add PBS so that the tubes are more than 2/3 full. If the tubes contain less liquid than that, the tubes will collapse during centrifugation! Tubes can be washed, autoclaved and reused several times.</li>
	<li>Pellet the exosomes through ultracentrifugation at 120,000 x g for 70 min at 4 &#176;C. After the centrifugation look at the bottom of the ultracentrifuge tube to sometimes see small yellow pellets; these are the exosomes.</li>
	<li>Discard the supernatant by carefully decanting it and disinfect the waste by adding bleach to a final concentration of 10% v/v and let it stand for 20 min.</li>
	<li>For downstream experiments, either process the pellet(s) in a small volume of the appropriate solution (i.e., radioimmunoprecipitation assay (RIPA) buffer for protein isolation, RNA lysis buffer for miR isolation) or resuspend it in 50-150 &#181;l of phosphate buffered saline (PBS) which can be either stored at -80 &#176;C for future use (quantitative real-time polymerase chain reaction (qRT-PCR) or protein extraction etc.) or used for characterization of the exosome preparation by TEM or nanoparticle optical analysis.</li>
</ol>
2. Exosome Identification by Transmission Electron Microscopy (TEM) or CryoEM
<ol>
	<li>Resuspend extracellular vesicles in 100 &#181;l of PBS by pipetting. Ideally, use exosomes that have not been stored at -20 &#176;C or -80 &#176;C, especially when performing the isolation for the first time.</li>
	<li>Adsorb a 20 &#181;l aliquot (about 2 &#181;g) to 400 mesh carbon-coated Parlodion copper grids for 2 min and allow to dry at RT.</li>
	<li>Fix the exosome in 1% (v/v) glutaraldehyde (EM-grade) for 5 min at RT by placing drops carefully on the dried preparation.</li>
	<li>Wash the grids twice with water for 5 min and then contrast stain EVs with 1% phosphotungstic acid (PTA) for 30 sec.</li>
	<li>Acquire images with an electron microscope with an acceleration voltage of 80 kilovolts starting at magnifications of 20,000X and increasing to 100,000X when determining the size of the particles. 
	Note: Other preps like layering onto Formvar carbon-coated 200 mesh copper or nickel grids and contrast staining like 1% or 2% uranyl acetate for 10-15 min are also possible.</li>
	<li>For CryoEM, mix a 20 &#181;l aliquot (about 2 &#181;g) with 60 &#181;l Protein G gold 10 nm (1:3 ratio). This will help to determine the exosome diameter.</li>
	<li>Transfer the samples onto glow-discharged C-flat holey carbon grids and remove excess liquid by blotting. Freeze the grids by dipping them immediately into liquid ethane.</li>
	<li>Mount the grids in a cryoholder in liquid nitrogen.</li>
	<li>Acquire the images at 200 kV with magnifications of 20,000X and 100,000X.</li>
</ol>
3. Exosome Identification by Multi-parameter Nanoparticle Optical Analysis
<ol>
	<li>Resuspend the isolated exosomes in 100 &#181;l of PBS by pipetting.</li>
	<li>Dilute exosomes in 600 &#181;l PBS at several different ratios (like 1:50, 1:100, 1:300, 1:600 and 1:1,200).</li>
	<li>Visualize the exosomes by laser light scattering with the appropriate instrument and software in real-time according to manufacturer&#39;s protocol20.</li>
	<li>Increase the level of the camera in capture mode until particles become visible, then adjust the focus to make the particles look smooth.</li>
	<li>While in standard mode, decrease the camera to the lowest level that the particles can still be seen, if necessary adjust camera shutter and gain to achieve this.</li>
	<li>Visually check to ensure 20-100 particles are visible on the screen, if not flush the unit and adjust the dilution in PBS.</li>
	<li>Then adjust the capture duration to between 30-60 sec. After the video is captured, adjust the processing parameters until all particles are identified on the screen and process the video file.</li>
</ol>
4. Exosome Characterization by Western Blot
<ol>
	<li>Lyse exosome pellets from 2-5 ml of bile in 100 &#181;l of ice-cold radioimmunoprecipitation assay (RIPA) buffer with a protease inhibitor by pipetting up and down thoroughly and mix lysates vigorously for 30 sec&#160;on a vortex to solubilize the proteins effectively.</li>
	<li>While most of the time not necessary, use pulse-sonication of the samples on ice with 2 pulses for 10 sec each to ensure full lysis.</li>
	<li>Pellet insoluble material by centrifugation at 15,000 x g at 4 &#176;C for 5 min and transfer supernatant to a clean tube.</li>
	<li>Determine the protein concentration with a method of choice (i.e., bicinchoninic acid (BCA), Coomassie, modified Lowry, 660 nm Protein Assay, etc.) according to manufacturer&#39;s protocol. The method is not as important as long as the same method is used consistently to achieve results that can be compared across experiments.</li>
	<li>Load 50 &#181;g of protein per sample in Laemmli buffer after heating the sample at 95 &#176;C for 5 min into a well of a polyacrylamide gel for electrophoresis (PAGE).</li>
	<li>Electrophorese the samples on the PAGE gels for 1.5 hr and transfer the separated proteins on to a nylon membrane.</li>
	<li>Block the membrane(s) with 5% nonfat milk in Tris-buffered Saline with 0.1% Tween 20 (TBS-T) for 1 hr, then incubate with exosome-specific antibodies like anti-CD63 and anti-TSG-101 at a dilution of 1:500 preferably O/N at 4 &#176;C.</li>
	<li>Wash the membranes with TBS-T 3 x 10 min, then incubate with an appropriate, matching HRP-conjugated secondary antibody in 5% nonfat milk in TBS-T for 1-2 hr at RT.</li>
	<li>Wash the membrane(s) 3 x 5 min with TBS-T and detect the specific proteins with chemiluminescence reagents according to manufacturer&#39;s protocol.</li>
	<li>Image the membranes with film or a digital imaging system according to manufacturer&#39;s protocol.</li>
</ol>
5. MicroRNA Isolation from the Isolated Exosomes
Note: Isolation of miRNA from bile exosomes is carried out using a modified miRNA isolation based on a commercially available kit, but any other RNA isolation method can be used or adapted.
<ol>
	<li>Add 300 &#181;l lysis/building buffer to the exosome pellet (isolated from 400 &#181;l bile), vortex, pipet or pass through a needle/syringe to completely lyse the exosomes to obtain a homogenous lysate.</li>
	<li>Add 1/10 (30 &#181;l) volume of miRNA Homogenate Additive, and mix well by vortexing or inverting the tube several time.</li>
	<li>Incubate the mix for 10 min on ice.</li>
	<li>Add a volume of acid-phenol:chloroform equal to the initial homogenate (300 &#181;l).</li>
	<li>Add 5 &#181;l of 5 fmol/&#181;l cel-miR 39 and vortex for 30-60 sec.</li>
	<li>Centrifuge for 5 min at 10,000 x g at RT to separate the aqueous (upper) and organic (lower) phases.</li>
	<li>Carefully aspirate the upper phase by pipetting and transfer to a fresh tube while noting the volume transferred. Discard the organic phase in the appropriate organic waste container.</li>
	<li>To enrich for small RNAs add 1/3 volume of 100% ethanol to the recovered aqueous phase and mix by thoroughly vortexing or inverting the tube several times.</li>
	<li>Pipet the lysate/ethanol mixture onto the filter cartridge, up to 700 &#181;l at a time.</li>
	<li>Centrifuge the cartridges for 15 sec at a maximum of 10,000 x g or apply vacuum to pass the mixture through the filter.</li>
	<li>Collect the filtrate and if the lysate/ethanol mixture exceeds 700 &#181;l, transfer the flow-through to a fresh tube and repeat the procedure to collect all the filtrates. These filter contains RNA fractions devoid of small RNAs and can be used to recover these larger RNAs.</li>
	<li>Add 2/3 volume of RT 100% ethanol to the total filtrate and mix thoroughly by vortexing.</li>
	<li>Pipet the filtrate/ethanol mixture onto a second filter cartridge. Like before the maximum volume that can be applied at once is 700 &#181;l.</li>
	<li>Centrifuge for 15 sec at 10,000 x g or apply vacuum as described in step 5.10.</li>
	<li>Discard the flow-through, and repeat until all of filtrate/ethanol mixture has been filtered.</li>
	<li>Apply 700 &#181;l miRNA Wash Solution 1 to the filter cartridge, centrifuge for 5-10 sec or apply vacuum and discard the flow-through.</li>
	<li>Wash the filter with 500 &#181;l wash solution 2/3 as done in step 5.16.</li>
	<li>Repeat the wash a second time with 500 &#181;l of wash solution 2/3 as in step 5.16, discard the flow-through and place the filter back in the collection tube.</li>
	<li>Spin the filter cartridge for 1 min at 10,000 x g to remove all residual fluid from the filter.</li>
	<li>Transfer the filter cartridge into a fresh collection tube and apply 100 &#181;l of hot (95 &#176;C) nuclease free water to the center of the filter.</li>
	<li>Spin the assemblies for 20-30 sec at maximum speed to recover the RNA.</li>
	<li>Collect the eluate and use immediately or store at -80 &#176;C.</li>
</ol>

<ol>
	<li>Admyre, 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=Exosomes+with+immune+modulatory+features+are+present+in+human+breast+milk.">Exosomes with immune modulatory features are present in human breast milk.</a> J. Immunol. 179 (3), 1969-1978 (2007).</li><li>Aushev, V. 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=Comparisons+of+microRNA+patterns+in+plasma+before+and+after+tumor+removal+reveal+new+biomarkers+of+lung+squamous+cell+carcinoma.">Comparisons of microRNA patterns in plasma before and after tumor removal reveal new biomarkers of lung squamous cell carcinoma.</a> PLoS One. 8 (10), e78649 (2013).</li><li>Ben-Dov, I. Z., 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=Urine+microRNA+as+potential+biomarkers+of+autosomal+dominant+polycystic+kidney+disease+progression:+description+of+miRNA+profiles+at+baseline.">Urine microRNA as potential biomarkers of autosomal dominant polycystic kidney disease progression: description of miRNA profiles at baseline.</a> PLoS One. 9 (1), e86856 (2014).</li><li>Vlassov, A. V., Magdaleno, S., Setterquist, R., Conrad, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+current+knowledge+of+their+composition,+biological+functions,+and+diagnostic+and+therapeutic+potentials.">Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials.</a> Biochim. Biophys. Acta. 1820 (7), 940-948 (2012).</li><li>Kosaka, N., Iguchi, H., Yoshioka, Y., Takeshita, F., Matsuki, Y., Ochiya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretory+mechanisms+and+intercellular+transfer+of+microRNAs+in+living+cells.">Secretory mechanisms and intercellular transfer of microRNAs in living cells.</a> J. Biol. Chem. 285 (23), 17442-17452 (2010).</li><li>Mittelbrunn, 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=Unidirectional+transfer+of+microRNA-loaded+exosomes+from+T+cells+to+antigen-presenting+cells.">Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells.</a> Nat. Commun. 2, 282 (2011).</li><li>Pegtel, D. M., van de Garde, M. D., Middeldorp, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+miRNAs+exploiting+the+endosomal-exosomal+pathway+for+intercellular+cross-talk+and+immune+evasion.">Viral miRNAs exploiting the endosomal-exosomal pathway for intercellular cross-talk and immune evasion.</a> Biochim. Biophys. Acta. 1809 (11-12), 715-721 (2011).</li><li>Peinado, H., 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=Melanoma+exosomes+educate+bone+marrow+progenitor+cells+toward+a+pro-metastatic+phenotype+through+MET.">Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.</a> Nat. Med. 18 (6), 883-891 (2012).</li><li>Thery, C., Ostrowski, M., Segura, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+vesicles+as+conveyors+of+immune+responses.">Membrane vesicles as conveyors of immune responses.</a> Nat. Rev. Immunol. 9 (8), 581-593 (2009).</li><li>Valadi, H., Ekstrom, K., Bossios, A., Sjostrand, M., Lee, J. J., Lotvall, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome-mediated+transfer+of+mRNAs+and+microRNAs+is+a+novel+mechanism+of+genetic+exchange+between+cells.">Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.</a> Nat. Cell Biol. 9 (6), 654-659 (2007).</li><li>Bessho, 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=Integrative+genomics+identifies+candidate+microRNAs+for+pathogenesis+of+experimental+biliary+atresia.">Integrative genomics identifies candidate microRNAs for pathogenesis of experimental biliary atresia.</a> BMC Syst. Biol. 7, (2013).</li><li>Calin, G. A., 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=MicroRNA+profiling+reveals+distinct+signatures+in+B+cell+chronic+lymphocytic+leukemias.">MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias.</a> Proc. Natl. Acad. Sci. U.S.A. 101 (32), 11755-11760 (2004).</li><li>Collins, A. 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=A+differential+microRNA+profile+distinguishes+cholangiocarcinoma+from+pancreatic+adenocarcinoma.">A differential microRNA profile distinguishes cholangiocarcinoma from pancreatic adenocarcinoma.</a> Ann. Surg. Oncol. 21 (1), 133-138 (2014).</li><li>He, 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=A+microRNA+polycistron+as+a+potential+human+oncogene.">A microRNA polycistron as a potential human oncogene.</a> Nature. 435 (7043), 828-833 (2005).</li><li>Taylor, D. D., Gercel-Taylor, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+signatures+of+tumor-derived+exosomes+as+diagnostic+biomarkers+of+ovarian+cancer.">MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer.</a> Gynecol. Oncol. 110 (1), 13-21 (2008).</li><li>Rabinowits, G., Gercel-Taylor, C., Day, J. M., Taylor, D. D., Kloecker, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomal+microRNA:+a+diagnostic+marker+for+lung+cancer.">Exosomal microRNA: a diagnostic marker for lung cancer.</a> Clin. Lung Cancer. 10 (1), 42-46 (2009).</li><li>Skog, 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=Glioblastoma+microvesicles+transport+RNA+and+proteins+that+promote+tumour+growth+and+provide+diagnostic+biomarkers.">Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.</a> Nat.Cell Biol. 10 (12), 1470-1476 (2008).</li><li>Lance, R. S., Drake, R. R., Troyer, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+recognition+assay+reveals+prostasomes+as+promising+plasma+biomarkers+for+prostate+cancer.">Multiple recognition assay reveals prostasomes as promising plasma biomarkers for prostate cancer.</a> Expert Rev. Anticancer Ther. 11 (9), 1341-1343 (2011).</li><li>Li, 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=Human+bile+contains+microRNA-laden+extracellular+vesicles+that+can+be+used+for+cholangiocarcinoma+diagnosis.">Human bile contains microRNA-laden extracellular vesicles that can be used for cholangiocarcinoma diagnosis.</a> Hepatology. 60 (3), 896-907 (2014).</li><li>Gabriel, D. A., Giordano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticle+sizing+and+counting+using+light+scattering+methods.">Microparticle sizing and counting using light scattering methods.</a> Semin. Thromb. Hemost. 36 (8), 824-832 (2010).</li><li>Colombo, M., Raposo, G., Thery, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis,+secretion,+and+intercellular+interactions+of+exosomes+and+other+extracellular+vesicles.">Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.</a> Annu. Rev. Cell Dev. Biol. 30, 255-289 (2014).</li></ol>

None of the authors have competing financial interests.

To reliably utilize exosomes isolated from bile, it is important to employ consistent high quality isolation methods to obtain high quality samples in return. The methodology defined in this paper is a well-established way to isolate exosomes and miRNA from human bile. It highlights several crucial steps in the characterization of the isolated exosomes which at a minimum should comprise electron microscopy or nanoparticle optical analysis and Western blots.
The most crucial step in the isolati...

Like other biological fluids, i.e., breast milk, plasma or urine, bile contains exosomes, lipid rich vesicles1-4. Exosomes can induce or alter biological functions in recipient cells5, 6, a form of cell-cell communication that might be part of their normal function4, 7-9. Exosomes can contain miRNA species which can provide a valuable source of biomarkers for diagnosis10. miRNAs profiles have gained substantial attention in recent years as diagnostic and prognostic markers for a variety of diseases11-14.
miRNA itself can be found in biological fluids, possibly released by dead cells and the amount of shed cells can be greatly influenced by an underlying disease. The miRNA profile of exosomes does not necessarily reflect the miRNA profile of the originating cell6, 10, 15-17, yet exosomes may carry miRNA signatures that might be characteristic for the parental cell like a tumor cell. Tumor-derived exosomes have been identified in the plasma of patients with lung adenocarcinoma, prostate cancer and other tumors8, 16, 18.
The goal of this method is the reliable and robust isolation of exosomes from human bile specimens, largely independent of their prior handling procedures. It was developed to utilize bile as a reliable source of miRNA for the potential diagnosis of diseases of the bile duct such as cholangiocarcinoma19. It consist of several steps of centrifugation with increasing speeds to isolate exosomes and might be applicable to other biological fluids.

<table border="0" cellpadding="0" cellspacing="0" width="752">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:305px;">0.2&#181;m Polyethersulfone (PES) membrane filter</td>
			<td style="width:161px;">Corning</td>
			<td align="right" style="width:119px;">431229</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">thinwall polyallomer ultracentrifuge tubes&#160;</td>
			<td style="width:161px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">331374</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">SW40Ti rotor</td>
			<td style="width:161px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">LM10-HS&#160;</td>
			<td style="width:161px;">Nanosight</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:305px;">Nanoparticle Tracking Analysis (NTA) software&#160;</td>
			<td style="width:161px;">Nanosight</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">mirVANA</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">AM1560</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:305px;">cOmplete Protease Inhibitor</td>
			<td style="width:161px;">Roche distributed by Sigma-Aldrich</td>
			<td align="right" style="width:119px;">4693116001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Immobilon PSQ</td>
			<td style="width:161px;">Millipore, Bedford MA</td>
			<td style="width:119px;">ISEQ00010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Anti-CD63 antibody</td>
			<td style="width:161px;">Abcam</td>
			<td style="width:119px;">ab59479</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Anti-Tsg101</td>
			<td style="width:161px;">Abcam</td>
			<td style="width:119px;">ab30871</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and profiling of microrna-containing exosomes from human bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Since exosomes are too small to be detected by regular microscopy or flow cytometry, electron microscopy or nanoparticle optical analysis has to be performed. The nanoparticle optical analysis has the advantage over electron microscopy that it is also quantitative and provides size distribution and concentration. The instrument introduces a finely focused laser beam through a glass prism into the sample. The Brownian motion of the isolated vesicles is captured via an EMCCD high sensitivit...

Watch this Scientific Journal Video about Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile at JoVE.com

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

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Medicine

10.2K Views.  Johns Hopkins University School of Medicine. Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Video: Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

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Isolation of Human Islets from Partially Pancreatectomized Patients

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

Isolation of Atrial Myocytes from Adult Mice

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping