This research was supported by internal funds from CSU (to KMD), ATCC contract #2016-0550-0002 (a subcontract of NIAID HHSN272201600013C), and The Bill and Melinda Gates Foundation (OPP1039688) (to KMD/NKG). We thank the NSF Research Experience for Undergraduates (REU) summer program at Colorado State University for additional support.

This work was determined not to be human subject research upon initial review from Colorado State University&#39;s Institutional Review Board (IRB), as all human samples were obtained as de-identified samples from the Bioreclamation IVT biorepository and were collected under IRB approved protocols.
1. Preparation of Crude Sample (either Plasma or Serum) by Pelleting Larger Vesicles and Cellular Debris
Note: Safety consideration: plasma, serum, and other biological fluids are biohazardous materials and must be handled with special care, while wearing basic personal protective equipment, including gloves and lab coat. It is highly recommended that biological samples are processed in a biosafety cabinet; if not available, the use of eye-protection is recommended.
<ol>
	<li>Equilibrate a serum or a plasma sample by placing the tube under non-shaking conditions in a 4 &#176;C refrigerator overnight (from either -20 or -80 &#176;C storage).</li>
	<li>Aliquot 250 &#181;L of the sample into a microcentrifuge tube using a 1,000 &#181;L pipette.</li>
	<li>Centrifuge the sample at 18,000 x g for 30 min at 4 &#176;C.</li>
	<li>Using a 200 &#181;L pipette, remove 200 &#181;L of the cleared supernatant and add it to a new microcentrifuge tube. Avoid any pelleted material while transferring the supernatant. Discard the pelleted material.</li>
	<li>Quantify the protein content of the sample by bicinchoninic acid (BCA) assay, using the manufacturer&#39;s protocol. To perform the assay, dilute the sample 1:50 in 1x phosphate-buffered saline (PBS). Test each sample in duplicate. 
	Note: Use 1x PBS throughout the protocol unless stated otherwise. On average, 200 &#181;L of clarified serum or plasma (after 18,000 x g) will yield 15 mg of total protein. Other sample types, with lower or higher protein content, may require additional BCA dilutions.</li>
	<li>Aliquot 10 mg of the sample into a new microcentrifuge tube. Use the concentration obtained in step 1.5 to calculate the corresponding volume of sample. Aliquot the sample using a 200 &#181;L pipette. 
	Note: It is recommended to store the remaining sample at -80 &#176;C. Aliquot the sample in different tubes at 10 mg per tube to avoid multiple freeze-thaw cycles in future use.</li>
</ol>
2. Digestion of Non-exosomal Blood Proteins
Note: The digestion step is designed to reduce the non-exosomal proteins which can co-purify with exosomes. If preservation of exosome surface proteins is required for downstream analyses, it should be taken into consideration that this step has the potential to interfere with this analysis. The detection of the exosomal CD63, a surface-exposed tetraspanin protein, was not significantly negatively impacted by this process.
<ol>
	<li>Prepare proteinase K solution at a concentration of 500 &#181;g/mL in PBS. Add the corresponding amount of proteinase K in a conical tube. Then add the buffer and vortex for 30 s, 3x at room temperature.</li>
	<li>Add 50 &#181;L of proteinase K solution to the 10-mg sample aliquot and mix gently by pipetting up and down using a 200 &#181;L pipette.</li>
	<li>Incubate the sample at 37 &#176;C in a water bath for 30 min. Place the tube in a float tube rack and avoid complete immersion of tube in the water bath to avoid potential leakage.</li>
	<li>Transfer the sample and floating tube rack to a 60 &#176;C water bath for 10 min, to inactivate the proteinase K.</li>
</ol>
3. Removal of Small Proteins and Peptides by Centrifuge Ultrafiltration
<ol>
	<li>Rinse an ultrafiltration device containing 100 kDa molecular weight cut-off (MWCO) filter with PBS prior to use. Do this by adding 500 &#181;L of PBS to the filter, using a 1,000 &#181;L pipette. Spin the device at 3,700 x g for 5 min. Discard any remaining retentate as well as the eluate. 
	Note: The sample volume capacity of the ultrafiltration device must be 0.5 mL - 4 mL to ensure that the final reduced sample volume of 50 &#181;L is achievable.</li>
	<li>Take the sample from step 2.4 and increase the volume to 500 &#181;L by adding PBS. Pipette the sample into the pre-rinsed ultrafiltration device. 
	Note: On average, the sample volume from step 2.4 is 185 &#181;L (120 to 150 &#181;L of sample and 50 &#181;L of proteinase K solution).</li>
	<li>Place the ultrafiltration device in a fixed-angle benchtop centrifuge at 3,700 x g at 4 &#176;C until the sample has reduce to a volume of 50 &#181;L. 
	CAUTION: When using ultrafiltration devices with dead stop volume, care must be taken to avoid complete dryness of the filter membrane during centrifugation steps.</li>
	<li>Add 500 &#181;L of PBS directly into the filter membrane and pipette up and down 5 times. Centrifuge as in step 3.3. Perform this wash step a total of 3 times.</li>
	<li>Transfer the final 50 &#181;L retentate in a new microcentrifuge tube.</li>
	<li>Rinse the filter membrane with 200 &#181;L PBS, pipetting up and down 10 times, and transfer the wash to the tube from step 3.5.</li>
	<li>Place the filter holder in the inverse position in a new tube. Run a reverse spin recovery for 5 min at 2,000 x g to retrieve remaining sample from the membrane. Pool the sample with the sample from step 3.5.</li>
</ol>
4. Purification of Vesicles by Size Exclusion Chromatography (SEC)
<ol>
	<li>Add 850 &#181;L of SEC slurry, with beads having a MWCO of 700 kDa, into an empty, capped gravity flow column using a 1,000 &#181;L pipette. Place the columns in a size appropriate rack fitted with a drip tray.</li>
	<li>Uncap the bottom of the column and let the liquid drain for 5 min. Once drained, add 10 mL of PBS to wash the resin. Allow 20 min for the wash to drain from the column.</li>
	<li>Place the concentrated, proteinase K treated sample from step 3.5 into a 15-mL conical tube, and increase the sample volume to 5 mL adding PBS with a serological pipette. Mix the tube gently by rocking for 1 min and then slowly apply the sample to the column prepared in step 4.2 with a serological pipette. 
	Note: Once the cap of the column is removed, the sample will flow through the resin by gravity; the 5-mL sample will completely flow through the column in 10 min.</li>
	<li>Collect the flow through in a new 15 mL conical tube.</li>
	<li>Using a serological pipette, apply the collected material to the resin again to remove any remaining material below 700 kDa.</li>
	<li>Collect the flow through in a new 15 mL conical tube. Wash the resin twice adding 1 mL of PBS. Collect the flow through in the tube from step 4.5; the total final volume will be roughly 7 mL. 
	Note: After step 4.6, the sample will be diluted about 35 times from the original sample volume; the following steps will reduce the volume and concentrate the purified exosome sample.</li>
</ol>
5. Concentration of Purified Exosomes and Total Protein Quantification
<ol>
	<li>Rinse an ultrafiltration device with a 3 kDa MWCO filter with PBS prior to use, as described in step 3.1.</li>
	<li>Add the sample from step 4.6 to the ultrafiltration device and centrifuge in a swinging-bucket rotor at 3,700 x g at 4 &#176;C. Buffer will pass through the filter and can be discarded. Concentrated exosomes will be retained above the filter. Centrifuge the sample until the volume above the filter (retentate) is reduced to 200 &#181;L.</li>
	<li>Transfer the retentate to a new microcentrifuge tube.</li>
	<li>Rinse the filter membrane with 200 &#181;L PBS by pipetting over the membrane 10 times and transfer the wash to the tube from step 5.3. This will allow for the collection of any residual exosome sample.</li>
	<li>Bring sample volume up to 500 &#181;L by adding PBS. Mix by slowly pipetting up and down 10 times. 
	Note: The protocol can be paused here. Samples should be stored at 4 &#176;C overnight or at -20/-80 &#176;C for long term storage.</li>
	<li>Quantify the protein content of the sample by BCA assay, using the manufacturer&#39;s protocol. Dilute the sample 1:10 and 1:50 in PBS. Run each sample in duplicate. 
	Note: Starting with 10 mg of total protein of serum or plasma, the total protein yield in purified exosomes from step 5.6 is, on average, 150 &#181;g. Additional dilutions may be necessary if samples other than serum or plasma are used; yield may vary with sample type.</li>
</ol>
6. Quantification and Sizing of Exosomes by Nanoparticle Tracking Analysis (NTA)
<ol>
	<li>Add 5 &#181;g of purified exosomes (as quantified in 5.6) to 1 mL of PBS and vortex for 15 s on low-medium speed.
	<ol>
		<li>Place the sample in a 1 mL disposable syringe. If available, set the syringe in an automatic syringe pump set to inject the diluted exosome sample at a rate of 30 &#181;L/min.</li>
		<li>Perform NTA measurements using video capture settings: screen gain of 3 - 4 and camera level of 12 - 13.</li>
		<li>Define the script for analysis to run three technical replicates for a minimum of 30 s using a constant flow for every replicate. For the analysis, set the capture threshold at 5. 
		Note: Details regarding the use of the automatic syringe, and settings for video capture are available in the reference13. Capture and analysis settings for NTA must be consistent across different samples to assure comparability.</li>
	</ol>
	</li>
</ol>
7. Determination of Soluble Protein Reduction
<ol>
	<li>Add 1 - 10 &#181;g of purified exosomes to SDS sample buffer and perform polyacrylamide gel electrophoresis (PAGE) using a 4 - 12% Bis-Tris Gel in 1x MES SDS running buffer for 35 min at 200 V. Transfer resolved proteins to a nitrocellulose 0.2 &#181;m membrane by applying a voltage of 50 V for 1 - 1.5 h. Perform western blot analysis to evaluate the presence of albumin, apolipoproteins A and B, and the exosome marker CD 63 following standard methodologies. 
	Note: Full protocols for the methods in 7.1 can be found in reference14; specifically, SP007: Running of polyacrylamide gels and SP011: Western blot protocol.</li>
	<li>Separate 10 &#181;g of purified exosomes using PAGE as in step 7.1 and stain the protein bands using coomassie dye, following standard recommendations, to visualize protein variation and reduction between samples. 
	Note: Further details regarding the protocol for western blots specific to CD63 are described by Diaz et al.15</li>
</ol>

<ol><li>Stoorvogel, W., Kleijmeer, M. J., Geuze, H. J., Raposo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+biogenesis+and+functions+of+exosomes%5BTitle%5D)+AND+%22Traffic%22%5BJournal%5D)">The biogenesis and functions of exosomes.</a> Traffic. 3 (5), 321-330 (2002).</li>
<li>Raposo, G., Stoorvogel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+vesicles%3A+exosomes%2C+microvesicles%2C+and+friends%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Extracellular vesicles: exosomes, microvesicles, and friends.</a> J Cell Biol. 200 (4), 373-383 (2013).</li>
<li>Schorey, J. S., Harding, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+vesicles+and+infectious+diseases%3A+new+complexity+to+an+old+story%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Extracellular vesicles and infectious diseases: new complexity to an old story.</a> J Clin Invest. 126 (4), 1181-1189 (2016).</li>
<li>Taylor, D. D., Gercel-Taylor, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exosome+platform+for+diagnosis+and+monitoring+of+traumatic+brain+injury%5BTitle%5D)+AND+%22Philos+Trans+R+Soc+Lond+B+Biol+Sci%22%5BJournal%5D)">Exosome platform for diagnosis and monitoring of traumatic brain injury.</a> Philos Trans R Soc Lond B Biol Sci. 369 (1652), (2014).</li>
<li>Nilsson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prostate+cancer-derived+urine+exosomes%3A+a+novel+approach+to+biomarkers+for+prostate+cancer%5BTitle%5D)+AND+%22Br+J+Cancer%22%5BJournal%5D)">Prostate cancer-derived urine exosomes: a novel approach to biomarkers for prostate cancer.</a> Br J Cancer. 100 (10), 1603-1607 (2009).</li>
<li>Linares, R., Tan, S., Gounou, C., Arraud, N., Brisson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-speed+centrifugation+induces+aggregation+of+extracellular+vesicles%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">High-speed centrifugation induces aggregation of extracellular vesicles.</a> J Extracell Vesicles. 4, 29509(2015).</li>
<li>Lobb, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimized+exosome+isolation+protocol+for+cell+culture+supernatant+and+human+plasma%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">Optimized exosome isolation protocol for cell culture supernatant and human plasma.</a> J Extracell Vesicles. 4, 27031(2015).</li>
<li>Vergauwen, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Confounding+factors+of+ultrafiltration+and+protein+analysis+in+extracellular+vesicle+research%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research.</a> Scientific Reports. 7 (1), 2704(2017).</li>
<li>Welton, J. L., Webber, J. P., Botos, L. A., Jones, M., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ready-made+chromatography+columns+for+extracellular+vesicle+isolation+from+plasma%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">Ready-made chromatography columns for extracellular vesicle isolation from plasma.</a> J Extracell Vesicles. 4, 27269(2015).</li>
<li>Sódar, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low-density+lipoprotein+mimics+blood+plasma-derived+exosomes+and+microvesicles+during+isolation+and+detection%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection.</a> Sci Rep. 6, 24316(2016).</li>
<li>de Menezes-Neto, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Size-exclusion+chromatography+as+a+stand-alone+methodology+identifies+novel+markers+in+mass+spectrometry+analyses+of+plasma-derived+vesicles+from+healthy+individuals%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">Size-exclusion chromatography as a stand-alone methodology identifies novel markers in mass spectrometry analyses of plasma-derived vesicles from healthy individuals.</a> J Extracell Vesicles. 4, 27378(2015).</li>
<li>Corso, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reproducible+and+scalable+purification+of+extracellular+vesicles+using+combined+bind-elute+and+size+exclusion+chromatography%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Reproducible and scalable purification of extracellular vesicles using combined bind-elute and size exclusion chromatography.</a> Scientific Reports. 7 (1), 11561(2017).</li>
<li>Nanosight Ltd. NanoSight NTA 2.1 Analytical Software, Operating Manual. , Available from:
 <a target="_blank" href="http://nanobio.physics.ucsb.edu/pdfs/equipment/Nanosight%20NTA2.1%20software%20manual.pdf">http://nanobio.physics.ucsb.edu/pdfs/equipment/Nanosight%20NTA2.1%20software%20manual.pdf</a> (2010).</li>
<li>Dobos, K. Production Manuals & SOPs. , Available from:
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<li>Diaz, G., Wolfe, L. M., Kruh-Garcia, N. A., Dobos, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+the+Membrane-Associated+Proteins+of+Exosomes+Released+from+Human+Macrophages+after+Mycobacterium+tuberculosis+Infection%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Changes in the Membrane-Associated Proteins of Exosomes Released from Human Macrophages after Mycobacterium tuberculosis Infection.</a> Sci Rep. 6, 37975(2016).</li>
<li>Yuana, Y., Levels, J., Grootemaat, A., Sturk, A., Nieuwland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Co-isolation+of+extracellular+vesicles+and+high-density+lipoproteins+using+density+gradient+ultracentrifugation%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">Co-isolation of extracellular vesicles and high-density lipoproteins using density gradient ultracentrifugation.</a> J Extracell Vesicles. 3, (2014).</li>
<li>Théry, C., Amigorena, S., Raposo, G., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+characterization+of+exosomes+from+cell+culture+supernatants+and+biological+fluids%5BTitle%5D)+AND+%22Curr+Protoc+Cell+Biol%22%5BJournal%5D)">Isolation and characterization of exosomes from cell culture supernatants and biological fluids.</a> Curr Protoc Cell Biol. , Chapter 3 Unit 3.22 (2006).</li>
<li>Muller, L., Hong, C. S., Stolz, D. B., Watkins, S. C., Whiteside, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+biologically-active+exosomes+from+human+plasma%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">Isolation of biologically-active exosomes from human plasma.</a> J Immunol Methods. 411, 55-65 (2014).</li>
<li>Böing, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-step+isolation+of+extracellular+vesicles+by+size-exclusion+chromatography%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">Single-step isolation of extracellular vesicles by size-exclusion chromatography.</a> J Extracell Vesicles. 3, (2014).</li>
<li>Sweeney, P. J., Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteinase+K+%28EC+3.4.21.14%29%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Proteinase K (EC 3.4.21.14).</a> Methods Mol Biol. 16, 305-311 (1993).</li>
<li>Baranyai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+Exosomes+from+Blood+Plasma%3A+Qualitative+and+Quantitative+Comparison+of+Ultracentrifugation+and+Size+Exclusion+Chromatography+Methods%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods.</a> PLoS One. 10 (12), e0145686(2015).</li>
</ol>

The authors have nothing to disclose.

An optimized method to increase the purity and yield of exosomes from blood will increase the ability to accurately mine extracellular vesicles as a source of biomarkers for several diseases. The standard methods currently used to isolate exosomes, specifically, ultracentrifugation and precipitation methods, have several disadvantages including exosome aggregation and pelleting of soluble proteins7,17,18. Alternatively, the use ...

Exosomes are nanovesicles (ranging in size from 30 nm to 150 nm) released by almost all the cells in the human body to facilitate cell-to-cell communication processes1,2. Interestingly, the composition of exosomes changes depending on the cells of origin as well as the health status of the individual3,4,5. Additionally, exosomes can be retrieved from several biological fluids such as: saliva, urine, and blood2. Because of these features, exosomes are considered to be a good source of disease biomarkers. Unfortunately, there is no standard method for the isolation of exosomes. Some laboratories consider multistep centrifugation, with a final high-speed step at 100,000 x g using density gradients as the gold standard method for exosome isolation. However, recent studies have shown that ultracentrifugation induces aggregation of exosomes with soluble proteins and other exosomes, in addition to affecting exosome integrity, both of which may hamper downstream applications6,7. Other common methods of exosome isolation include, but are not limited to: precipitation by commercial polyethylene glycol (PEG) based reagents, centrifugal ultrafiltration, and size-exclusion chromatography (SEC). The commercial reagents using polyethylene glycol (PEG) polymers enrich exosomes by causing them to precipitate and form a pellet. Limitations using this polymer are contamination with residual PEG polymer and an abundance of soluble non-exosomal proteins in the final product. Ultrafiltration utilizes centrifugation to purify and concentrate vesicles using a cellulose membrane; exosomes are retained above the filter, while smaller impurities and other proteins pass through the membrane7,8. Just like other methods, centrifugal ultrafiltration has a limited capacity to purify exosomes due to the retention of high levels of non-exosomal proteins, including protein complexes and aggregates. Finally, SEC purification uses porous resin to separate molecules by size. SEC has shown promising results, overcoming most of the problems experienced with other methods by capturing the majority of contaminant proteins and preserving exosomal integrity, since isolation is based on gravity or low-pressure systems7,9. However, the co-isolation of larger protein aggregates and lipoproteins10 during SEC affects the purity of the final exosome preparation. While some methods have been tested for exosome purification from cell culture supernatants and plasma7, or only plasma9,11, there is no information about the performance of methods directly comparing blood plasma and serum from the same individual.
Here, we focus on the purification of blood exosomes by comparing a variety of workflows to determine if vesicle enrichment techniques are translatable between plasma and serum. Nanoparticle tracking analysis and western blot were used to quantify the differences in exosome concentration, purity, and protein composition in the end products. The final method, detailed in this protocol, increases vesicle numbers and reduces non-exosomal protein levels. Importantly, a drastic reduction of common co-precipitating proteins including albumin and apolipoproteins is demonstrated. The high abundance of these two proteins in blood and the frequency at which they co-purify with exosomes causes inconsistencies between &#34;purified&#34; samples, skewing the downstream analyses. This protocol includes the use of a commercially available SEC resin; the resin is composed of porous beads in which proteins smaller than 700 kDa can enter the beads. Once inside, the proteins are retained by an octylamine ligand. The eluate is composed of exosomes and molecules larger than 700 kDa12. Additionally, in order to reduce the protein aggregates and apolipoproteins which evade bead trapping, we include a protease digestion step in the workflow. Currently, there is no single technique capable of maximizing exosome yield while reducing co-purifying non-exosomal proteins. This study shows that a purification protocol that combines a protease digestion pretreatment with multiple recovery and purification methods can be used to increase exosome yield and purity from blood serum and plasma.

<table><tbody><tr><td>Nanosight</td><td>Malvern</td><td>NS300</td><td></td></tr><tr><td>Benchtop microcentrifuge&amp;nbsp;</td><td>Thermo</td><td>Legend Mico21</td><td></td></tr><tr><td>Benchtop centrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>Allegra 6R</td><td></td></tr><tr><td>Waterbath&amp;nbsp;</td><td>Benchmark</td><td>B2000-4</td><td></td></tr><tr><td>Microplate reader</td><td>BIOTEK</td><td>Epoch</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>THERMO</td><td>23227</td><td></td></tr><tr><td>Micro BCA Protein Assay Kit</td><td>THERMO</td><td>23235</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>96 well plate</td><td>Corning</td><td>15705-066</td><td></td></tr><tr><td>Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-100 membrane</td><td>EMD-Millipore</td><td>UFC510096</td><td></td></tr><tr><td>Amicon Ultra-4 Centrifugal Filter Units</td><td>EMD-Millipore</td><td>UFC800324</td><td></td></tr><tr><td>Capto Core 700</td><td>GE Healthcare</td><td>17548103</td><td></td></tr><tr><td>Poly-Prep&amp;nbsp;Chromatography Columns</td><td>BIORAD</td><td>7311550</td><td></td></tr><tr><td>NuPAGE 4-12% Bis-Tris Protein Gels</td><td>THERMO</td><td>NP0323BOX</td><td></td></tr><tr><td>Nitrocellulose Membrane, Roll, 0.2 &amp;micro;m</td><td>BIORAD</td><td>1620112</td><td></td></tr><tr><td>Proteinase K,&amp;nbsp;&lt;em&gt;Tritirachium album&lt;/em&gt;</td><td>EMD-Millipore</td><td>539480</td><td></td></tr><tr><td>SimplyBlue SafeStain</td><td>THERMO</td><td>LC6065</td><td></td></tr><tr><td>SIGMAFAST&amp;nbsp;BCIP/NBT</td><td>Millipore-SIGMA</td><td>B5655</td><td></td></tr><tr><td>4-Chloro-1-naphthol</td><td>Millipore-SIGMA</td><td>C6788</td><td></td></tr><tr><td>Anti-CD63 Antibody (rabbit anti-human) with goat anti-rabbit HRP secondary antibody</td><td>SYSTEM BIOSCIENCES</td><td>EXOAB-CD63A-1</td><td>Primary and secondary antibodies</td></tr><tr><td>ALB Antibody (F-10)</td><td>SANTA CRUZ BIOTECHNOLOGY, INC.</td><td>sc-271605</td><td>Primary antibody</td></tr><tr><td>apoB Antibody (C1.4)</td><td>SANTA CRUZ BIOTECHNOLOGY, INC.</td><td>sc-13538</td><td>Primary antibody</td></tr><tr><td>apoA-I Antibody (B-10)</td><td>SANTA CRUZ BIOTECHNOLOGY, INC.</td><td>sc-376818</td><td>Primary antibody</td></tr></tbody></table>

protein digestion, ultrafiltration, and size exclusion chromatography to optimize the isolation of exosomes from human blood plasma and serum

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

The data presented below were obtained using paired plasma and serum samples from five healthy blood donors. To determine the effects of each processing step, five variations of the presented protocol were performed, and exosome recovery and non-exosome protein removal were compared. The methods trialed included: 1) SEC 700 kDa + Ultrafiltration 3 kDa; 2) Ultrafiltration 100 kDa; 3) Proteinase K + Ultrafiltration 100 kDa; 4) SEC 700 kDa + Ultrafiltration 100 kDa, and 5) Proteinase K + Ult...

Watch this Scientific Journal Video about Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum at JoVE.com

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

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

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

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Research

JoVE Journal

Biochemistry

16.5K Views.  Colorado State University. Hier presenteren we een protocol om te zuiveren van exosomes van plasma en serum met verminderde CO zuivering van niet-exosomal bloedeiwitten. Het geoptimaliseerde protocol bevat ultrafiltratie, protease behandeling en grootte uitsluiting chromatografie. Verbeterde zuivering van exosomes voordelen downstream analyses, met inbegrip van nauwkeuriger kwantificering van blaasjes en proteomic karakterisering.

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

Purification and Transplantation of Myogenic Progenitor Cell Derived Exosomes to Improve Cardiac Function in Duchenne Muscular Dystrophic Mice

Duchene Muscular Dystrophy (DMD) is an X-linked recessive genetic disease caused by a lack of functional dystrophin protein. The disease cannot be cured, and as the disease progresses, the patient develops symptoms of dilated cardiomyopathy, arrhythmia, and congestive heart failure. The DMDMDX mutant mice do not express dystrophin, and are commonly used as a mouse model of DMD. In our recent study, we observed that intramyocardial injection of wide type (WT)-myogenic progenitor cells-derived exosomes (MPC-Exo) transiently restored the expression of dystrophin in the myocardium of DMDMDX mutant mice, which was associated with a transient improvement in cardiac function suggesting that WT-MPC-Exo may provide an option to relieve the cardiac symptoms of DMD. This article describes the technique of MPC-Exo purification and transplantation into hearts of DMDMDX mutant mice.

Here, we present a protocol to transiently improve cardiac function in Duchenne muscular dystrophy mice by transplanting exosomes derived from normal myogenic progenitor cells.

Duchene Muscular Dystrophy (DMD) is an X-linked recessive genetic disease caused by a lack of functional dystrophin protein. The disease cannot be cured, ...

Genetics

A Modified Precipitation Method to Isolate Urinary Exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

Medicine

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.

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 ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Biology

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

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

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

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

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

Neuroscience

Refining Techniques for Murine Spleen-Derived Exosome Isolation

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed that Exo derived from humoral or cell-supernatant are promising targets for novel diagnostic or prognostic biomarkers, underscoring their significant role in disease pathogenesis. Tissue-derived Exo (Ti-Exo) have attracted increasing attention due to its ability to accurately reflect tissue specificity and the microenvironment. Ti-Exo, present in interstitial space, play crucial roles in intercellular communication and cross-organ signaling. Despite their recognized value in elucidating disease mechanisms, isolating Ti-Exo remains challenging due to the complexity of tissue matrices and variability in extraction methods. In this study, we developed a practical protocol for isolating exosomes from mice spleen tissue, providing a reproducible technique for subsequent identification analysis and functional studies. We used Type I collagenase digestion combined with differential ultracentrifugation to isolate spleen-derived Exo. The characteristics of isolated Exo were determined through electron microscopy, the nano-flow cytometer, and the western blot. The isolated spleen-derived Exo displayed the typical morphology of lipid bilayer vesicles, with particle sizes ranging from 30 nm to 150 nm. In addition, the expression profile of exosome markers confirmed the presence and purity of exosomes. Taken together, we successfully established a practical protocol for isolating spleen-derived Exo in mice.

Here, we present a protocol to isolate mouse spleen-derived exosomes using a combination of digestion with collagenase type I and ultracentrifugation.

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed ...

Cardiovascular Biomarker Studies Using Plasma Extracellular Vesicle Proteome

Isolation, Characterization, and Proteomic Analysis of Plasma-Derived Extracellular Vesicles for Cardiovascular Biomarker Discovery

Extracellular vesicles (EV) are cell-derived, lipid bilayer-enclosed, non-replicable nanoparticles. EV currently gain attention in cardiovascular research due to their role in regulating intercellular communication, potentially serving as valuable biomarkers for cardiovascular disease. However, the EV proteome and its potential as a biomarker in cardiovascular diagnostics remain poorly understood. This protocol presents a standardized method for the isolation and quantification of plasma-derived EV and the analysis of their protein cargo using plasma samples from patients presenting to the Chest Pain Unit of a large university hospital. Following routine phlebotomy, EV are isolated from plasma by differential ultracentrifugation. The enrichment of specific EV marker proteins in EV isolates is visualized by immunoblotting, and average size distribution and plasma EV concentrations are quantified by nanoparticle tracking analysis. Finally, ultra-performance liquid chromatography-tandem mass spectrometry is employed for label-free analysis of the EV proteome. This protocol thus provides a comprehensive approach to study and use plasma-derived EV as potential carriers of critical biological information as well as to explore their potential as novel biomarkers.

Author Spotlight: Advancing the Analysis of Plasma Extracellular Vesicle Proteome for Cardiovascular Biomarker Studies

This article describes a methodology for the isolation, characterization, and quantification of human plasma-derived extracellular vesicles (EV) and presents a workflow for label-free analysis of the EV proteome using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Extracellular vesicles (EV) are cell-derived, lipid bilayer-enclosed, non-replicable nanoparticles. EV currently gain attention in cardiovascular research ...

Magnetic Bead-based Exosome Extraction: A Technique to Isolate Specific Exosomes from Biofluids

Source: Tu, M. et al. <a href="https://www.jove.com/video/52439" target="_blank">Detection of Exosomal Biomarkers by Electric Field-induced Release and Measurement (EFIRM).</a> J. Vis. Exp. (2015)
In this video, we demonstrate a procedure to isolate exosomes from target biofluids using magnetic microbeads. The procedure enables the rapid capture of exosomes, which can be used for downstream analysis.

Source: Tu, M. et al. Detection of Exosomal Biomarkers by Electric Field-induced Release and Measurement (EFIRM). J. Vis. Exp. (2015)
In this video, we ...

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

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