This study was, in part or in whole, supported by National Natural Science Foundation of China (31472187 and 31672550) and The Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences.

All animal experiments were approved by the local ethics committee of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Permit Number: SHVRIAU-14-0101).
1. In Vitro Culture of Schistosomes
Note:&#160;Schistosomes represent a biohazard. Workers should wear latex gloves at all times when handling worms, schistosomal suspensions, or other related biological materials. New Zealand rabbits were infected with ~2,000 cercariae via abdominal exposure. Schistosomes at the liver stage were collected from rabbits infected with S. japonicum cercariae at 28 days post-infection by perfusion of liver and mesenteric veins. The procedures of worm collection have been referenced in previous publications reporting studies in mice18.
<ol>
	<li>Collect parasites in a 500-mL beaker. Wash them thoroughly and gently 3x with 200 mL of preheated (37 &#176;C) PBS (pH 7.4).</li>
	<li>Separate ~200 worm pairs for each 90 mm Petri dish. Then wash the parasites thoroughly and gently 2x with 50 mL of preheated (37 &#176;C) RPMI-1640 culture medium containing 100 U of penicillin and 100 mg/mL of streptomycin.</li>
	<li>Maintain parasites in 40 mL of preheated RPMI-1640 culture medium without antibiotics&#160;at 37 &#176;C under 5% CO2 at a density of ~5 worm pairs /mL for 2 h in 90 mm Petri dish. 
	Notes: The RPMI 1640 medium should not contain serum, otherwise exogenous extracellular vesicle will be introduced.</li>
</ol>
2. Pre-treated Condition Media
<ol>
	<li>Collect the culture medium and then centrifuge the medium at 4 &#176;C at 2,000 &#215; g for 30 min to remove eggs and worm tegmental debris.</li>
	<li>Transfer the supernatant to a fresh tube and then filter the solution using a 0.22 &#181;m syringe filter.</li>
	<li>Collect the filtered solution in a dialyzed bag (molecular weight cut-off: 3.5kD) and then dialyze the solution using 8% PEG8000 solution at 4 &#176;C overnight. 
	Notes: This step is important to enrich SjEVs for increasing the yield of SjEVs isolation.</li>
</ol>
3. Isolation of SjEVs
<ol>
	<li>Transfer the dialyzed solution to a new tube and then add 0.5 volumes of the total exosome isolation reagent.</li>
	<li>Mix the solution well by vortexing or pipetting up and down to ensure homogeneity of the solution.</li>
	<li>Incubate the mixture at 2 &#176;C to 8 &#176;C overnight.</li>
	<li>Centrifuge the mixture at 15,000 &#215; g for 1 h at 2 &#176;C to 8 &#176;C.</li>
	<li>Aspirate and discard the supernatant. 
	Notes: EVs are contained in the pellet at the bottom of the tube (not visible in most of the cases).</li>
	<li>Resuspend the EVs pellet in 2 mL of PBS and then store the EVs solution at -80 &#176;C until further analysis.</li>
</ol>
4. Characterization of EVs by Western Blotting
<ol>
	<li>Determine the protein concentration of the SjEVs sample, e.g., by BCA assay.</li>
	<li>Prepare 10 - 20 &#181;g of EVs in 22.5 &#181;L RIPA buffer. Then add 7.5 &#181;L of 4x loading buffer and heat for 5 min at 95 &#176;C.</li>
	<li>Separate SjEVs proteins by 12% SDS-PAGE gel.</li>
	<li>Transfer the proteins to PVDF membrane and then probe with anti-CD63 antibodies (1:1,000 dilution), followed by probing with anti-rabbit IgG-HRP (1:5,000 dilution).</li>
	<li>Use ECL detection reagent for membrane development and detect the signals by a chemiluminescence imaging system.</li>
</ol>
5. Characterization of EVs by Transmission Electron Microscopy
<ol>
	<li>Add 2 &#181;L of isolated SjEVs to 200 mesh formvar-coated grids and then allow it to dry at room temperature.</li>
	<li>Stain with 2% Phosphotungstic Acid for 1 min and then allow it to dry at room temperature.</li>
	<li>Load the grids onto the sample holder of the transmission electron microscope and exposed to an 80 kV electron beam for image capture.</li>
</ol>
6. SjEVs Label with PKH67 and Cell Uptake Assay
Notes: All subsequent steps were performed at ambient temperature (20 - 25 &#176;C), unless otherwise indicated.
<ol>
	<li>Transfer 5 &#181;g SjEVs solution into a conical bottom polypropylene tube and adjust the volume to 12 mL by adding RPMI 1640 medium.</li>
	<li>Centrifuge the SjEVs at 110,000 &#215; g for 90 min at 4 &#176;C to pellet SjEVs.</li>
	<li>After centrifugation, carefully aspirate the supernatant. 
	Note: The PKH67 ethanolic dye should not be added directly to the SjEV pellet, as this would result in heterogeneous staining and reduced cell viability.</li>
	<li>Prepare a 2x&#160;EV Suspension solution by adding 1 mL of Diluent C to the SjEV pellet; to ensure complete dispersion, resuspend with gentle pipetting.</li>
	<li>Just before staining in Diluent C, prepare a fresh 2x&#160;Dye Solution by adding 2 &#181;L of the PKH67 ethanolic dye solution to 1 mL of Diluent C in a polypropylene centrifuge tube and mixing well to disperse.</li>
	<li>Rapidly add the 1 mL of 2x EV Suspension (Step 6.4) to 1 mL of 2x&#160;Dye Solution (Step 6.5) and immediately mix the sample by pipetting.</li>
	<li>Incubate the mixture at room temperature for 4 min.</li>
	<li>Stop the staining by adding 4&#160;mL of FBS and then incubate for 1 min.</li>
	<li>Add RPMI1640 medium to adjust the volume to 12 mL and then centrifuge at 110,000 &#215; g at 4 &#176;C for 90 min.</li>
	<li>Remove the supernatant and add RPMI1640 medium to resuspend the PKH67 labeled SjEV pellets.</li>
	<li>Centrifuge at 110,000 &#215; g at 4 &#176;C for 90 min to wash once and then add PBS to resuspend the SjEVs pellets.</li>
</ol>
7. SjEVs Cell Uptake Assay
<ol>
	<li>Seed mammalian cells such as NCTC clone 1469 cells or HEK293T cells in 6-well plates (2&#215; 105 cells per well) and culture the cells overnight.</li>
	<li>Add 5 &#181;g PKH67-labelled SjEVs and then culture cells at 37 &#176;C for 2 - 4 h.</li>
	<li>After incubation, remove the medium and wash the cells with PBS twice.</li>
	<li>Fix the cells with 4% formaldehyde solution for 15 min and washed twice more with PBS.</li>
	<li>Stain cell nuclei with 4&#8242;,6-diamidino-2-phenylindole (DAPI).</li>
	<li>Observe the cells using fluorescence microscopy.</li>
</ol>

<ol>
	<li>Tetta, C., Ghigo, E., Silengo, L., Deregibus, M. C., Camussi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+as+an+emerging+mechanism+of+cell-to-cell+communication.">Extracellular vesicles as an emerging mechanism of cell-to-cell communication.</a> Endocrine. 44 (1), 11-19 (2013).</li><li>Abels, E. R., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+Extracellular+Vesicles:+Biogenesis,+RNA+Cargo+Selection,+Content,+Release,+and+Uptake.">Introduction to Extracellular Vesicles: Biogenesis, RNA Cargo Selection, Content, Release, and Uptake.</a> Cell Mol Neurobiol. 36 (3), 301-312 (2016).</li><li>Riaz, F., Cheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome-like+vesicles+of+helminths:+implication+of+pathogenesis+and+vaccine+development.">Exosome-like vesicles of helminths: implication of pathogenesis and vaccine development.</a> Ann Transl Med. 5 (7), 175 (2017).</li><li>Zhang, H. G., Grizzle, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+and+cancer:+a+newly+described+pathway+of+immune+suppression.">Exosomes and cancer: a newly described pathway of immune suppression.</a> Clin Cancer Res. 17 (5), 959-964 (2011).</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><li>Li, Y., 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=Circular+RNA+is+enriched+and+stable+in+exosomes:+a+promising+biomarker+for+cancer+diagnosis.">Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis.</a> Cell Res. 25 (8), 981-984 (2015).</li><li>Marcilla, 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=Extracellular+vesicles+in+parasitic+diseases.">Extracellular vesicles in parasitic diseases.</a> J Extracell Vesicles. 3, 25040 (2014).</li><li>Cwiklinski, 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=The+Extracellular+Vesicles+of+the+Helminth+Pathogen,+Fasciola+hepatica:+Biogenesis+Pathways+and+Cargo+Molecules+Involved+in+Parasite+Pathogenesis.">The Extracellular Vesicles of the Helminth Pathogen, Fasciola hepatica: Biogenesis Pathways and Cargo Molecules Involved in Parasite Pathogenesis.</a> Mol Cell Proteomics. 14 (12), 3258-3273 (2015).</li><li>Buck, A. 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=Exosomes+secreted+by+nematode+parasites+transfer+small+RNAs+to+mammalian+cells+and+modulate+innate+immunity.">Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity.</a> Nat Commun. 5, 5488 (2014).</li><li>Sotillo, 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=Extracellular+vesicles+secreted+by+Schistosoma+mansoni+contain+protein+vaccine+candidates.">Extracellular vesicles secreted by Schistosoma mansoni contain protein vaccine candidates.</a> Int J Parasitol. 46 (1), 1-5 (2016).</li><li>Nowacki, F. 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=Protein+and+small+non-coding+RNA-enriched+extracellular+vesicles+are+released+by+the+pathogenic+blood+fluke+Schistosoma+mansoni.">Protein and small non-coding RNA-enriched extracellular vesicles are released by the pathogenic blood fluke Schistosoma mansoni.</a> J Extracell Vesicles. 4, 28665 (2015).</li><li>Wang, 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=Exosome-like+vesicles+derived+by+Schistosoma+japonicum+adult+worms+mediates+M1+type+immune-+activity+of+macrophage.">Exosome-like vesicles derived by Schistosoma japonicum adult worms mediates M1 type immune- activity of macrophage.</a> Parasitol Res. 114 (5), 1865-1873 (2015).</li><li>Koh, Y. Q., Almughlliq, F. B., Vaswani, K., Peiris, H. N., Mitchell, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome+enrichment+by+ultracentrifugation+and+size+exclusion+chromatography.">Exosome enrichment by ultracentrifugation and size exclusion chromatography.</a> Front Biosci (Landmark Ed). 23, 865-874 (2018).</li><li>Safdar, A., Saleem, A., Tarnopolsky, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+endurance+exercise-derived+exosomes+to+treat+metabolic+diseases.">The potential of endurance exercise-derived exosomes to treat metabolic diseases.</a> Nat Rev Endocrinol. 12 (9), 504-517 (2016).</li><li>Livshits, M. 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=Isolation+of+exosomes+by+differential+centrifugation:+Theoretical+analysis+of+a+commonly+used+protocol.">Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol.</a> Sci Rep. 5, 17319 (2015).</li><li>Zhu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+of+extracellular+vesicles+containing+small+RNAs+from+the+eggs+of+Schistosoma+japonicum.">Release of extracellular vesicles containing small RNAs from the eggs of Schistosoma japonicum.</a> Parasit Vectors. 9 (1), 574 (2016).</li><li>Zhu, 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=Molecular+characterization+of+S.+japonicum+exosome-like+vesicles+reveals+their+regulatory+roles+in+parasite-host+interactions.">Molecular characterization of S. japonicum exosome-like vesicles reveals their regulatory roles in parasite-host interactions.</a> Sci Rep. 6, 25885 (2016).</li><li>Lewis, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> Curr Protoc Immunol. , 11 (2001).</li><li>Cheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+miRNAs:+roles+in+cancer+diagnosis,+prognosis+and+therapy.">Circulating miRNAs: roles in cancer diagnosis, prognosis and therapy.</a> Adv Drug Deliv Rev. 81, 75-93 (2015).</li></ol>

The authors have nothing to disclose.

Recent studies on EVs have demonstrated that schistosome EVs play an important role in host-pathogen interactions3,9,12,16. To further address their regulatory functions, it is essential to isolate EVs from schistosomes. Here, we describe an alternative method for SjEV isolation. This method yields a wide range size of SjEVs, from 100 nm to 400 nm, in adult S. japonicum. The following cell uptake assay indicated that EVs isolated under the described protocol were successfully internalized by the recipient cells and that their associated miRNAs potentially play regulatory roles17.
EVs, which may be secreted by many different types of cells, are also present in various kinds of body fluids19. Consequently, it is important to avoid to artificially introducing exogenous EVs during the process of SjEV isolation. For example, the culture medium should not contain serum. The worm density in the in vitro culture should be reasonable to minimize the potential stress response and lower the mortality rates of worms. In addition, it should be noted that different stages of schistosomes or worms from different hosts may present different EVs. Consequently, it is imperative to maintain the parasites in in vitro environments that closely resemble the host environment, and the conditions under which SjEV isolation is performed must be consistent. Additionally, we are unable to rule out the possibility of variance related to SjEVs caused by culture stress.
Here, we present a protocol for the isolation of SjEVs from 2 h in vitro culture media for adult schistosomes. An advantage of this protocol is that short culture time minimizes the stress to which parasites are exposed, which may result in non-physiological EV secretion. In addition, we found that the SjEVs isolated as described in this protocol were internalized by the recipient cells, suggesting that these SjEVs may be biologically active17. Overall, the present protocol allows the efficient isolation of SjEVs from adult worms using standard laboratory equipment, and enables their characterization using western blotting and cell uptake assay. The isolated EVs may be used to further characterize host-pathogen interactions.

Extracellular vesicles (EVs) are a population of small membrane-bound vesicles encapsulated with various proteins, lipids, and nucleic acids. Recent studies demonstrated that EVs play a crucial role in cell-cell communication, and are involved in the regulation of numerous physiological processes, including cell development, immune regulation, angiogenesis, and cell migration2,3,4,5. Accumulating evidence indicates that EVs, circulating exosomes, and their miRNA cargo represents potential biomarkers of certain diseases6.
Several protozoa such as Trichomonas vaginalis, Trypanosoma cruzi, and Leishmania spp., have been shown to be able to secrete EVs; helminths have additionally been found to secrete EVs into living hosts7. Parasitic EVs have been shown to be involved in the maintenance of infection, pathogenicity8, and immune regulation9. Recent studies in schistosomes both Schistosoma mansoni (S. mansoni) 10,11&#160;and S. japonicum12 have indicated that adult schistosomes secrete exosome-like vesicles that may be involved in the functional regulations of specific biological processes.
To date, several methods have been used to isolate extracellular vesicles, such as ultracentrifugation,ultrafiltration13, the use of polymer-based reagents, size-exclusion chromatography, and immunoaffinity isolation. These different methods possess their own advantages and limitations14. Generally, ultracentrifugation is considered the gold standard method for vesicle isolation. However, this method suffers from the limitation of potential EV aggregation14.
In schistosomes, a couple of methods have been reported for EV isolation: these include ultracentrifugation11,12,10 and the use of commercial EV isolation kit16 for several stages (eggs16, schistosomula11, adult schistosomes10,12,17). Given that microvesicles are of a wide range of size from several hundred nanometers to thousand nanometers, we developed an alternative method that combines with the use of bench centrifuge, ultrafiltration, and a commercial EV isolation kit to isolate the EVs from adult Schistosoma japonicum. The isolated EVs typically possessed a diameter of 100 - 400 nm and were successfully internalized by the recipient cells.

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Total Exosome Isolation Reagent (from cell culture media)</td><td>ThermoFisher SCIENTIFIC</td><td>4478359</td><td>For isolating S. japonicum extracellular vesicles</td></tr><tr><td>PKH67</td><td>Sigma-aldrich</td><td>MINI67</td><td>For labeling Evs</td></tr><tr><td>RPMI 1640 Medium</td><td>ThermoFisher SCIENTIFIC</td><td>11875119</td><td>For parasite culture</td></tr><tr><td>Penicillin-Streptomycin</td><td>ThermoFisher SCIENTIFIC</td><td>15140122</td><td></td></tr><tr><td>0.22&amp;mu;m syringe filter</td><td>Merck</td><td>SLGP033RB</td><td></td></tr><tr><td>SnakeSkin Dialysis Tubing</td><td>Thermo SCIENTIFIC</td><td>68035</td><td></td></tr><tr><td>PEG8000</td><td>Sangon Biotech</td><td>A100159&amp;nbsp;</td><td></td></tr><tr><td>RIPA</td><td>Beyotime</td><td>P0013B</td><td></td></tr><tr><td>EMD Millipore&amp;trade; Immobilon&amp;trade; Western Chemiluminescent HRP Substrate (ECL)</td><td>Fisher scientific</td><td>WBKLS0100&amp;nbsp;</td><td></td></tr><tr><td>DAPI</td><td>Cell Signaling Technology</td><td>4083</td><td></td></tr><tr><td>BCA kit</td><td>Beyotime</td><td>P0010</td><td></td></tr><tr><td>CD63 antibodies</td><td>Sangon Biotech</td><td>D160973-0025</td><td></td></tr><tr><td>PVDF</td><td>Bio-Rad</td><td>1704273</td><td></td></tr><tr><td>Formvar/Carbon 400 mesh</td><td>Ted Pella</td><td>01754-F</td><td></td></tr><tr><td>Phosphotungstic Acid</td><td>Ted Pella</td><td>19402</td><td></td></tr><tr><td>anti-mouse IgG-HRP</td><td>CWBIO</td><td>CW0102</td><td></td></tr><tr><td>NCTC clone 1469 cells</td><td>ATCC</td><td>ATCC&amp;reg; CCL-9.1&amp;trade;</td><td></td></tr><tr><td>FBS</td><td>HyClone</td><td>SV30087.02</td><td></td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GE chemoluminescance imaging system</td><td>GE</td><td>ImageQuant LAS4000mini</td><td></td></tr><tr><td>Transmission electron microscopy</td><td>Hitachi</td><td>H-7600</td><td></td></tr><tr><td>Milli-Q water</td><td>Milli-Q</td><td>Milli-Q Elix</td><td></td></tr><tr><td>Eppendor Centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>Centrifuge 5804R</td><td></td></tr><tr><td>Zetasizer Nano</td><td>Malvern</td><td>Zetasizer Nano ZS&amp;nbsp;</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman</td><td>Optima L-100 XP Ultracentrifuge</td><td></td></tr><tr><td>Incubator</td><td>ESCO</td><td>CCL-107B</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Zeiss Axin Observer Z1</td><td></td></tr></tbody></table>

isolation and characterization of extracellular vesicles from adult schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

To quantify the yield of isolated SjEVs using the described protocol, we used BCA protein assay to access the protein concentration of the isolated SjEVs from 28-day adult schistosomes. As shown in Table 1, the SjEV protein concentration ranged from 208 &#181;g to 250 &#181;g per 100 mL of medium (Table 1).The particle size, as determined by Malvern nanoparticle analysis, indicated that the isolated SjEVs ranged from 100 nm to 400 nm, with the highest population around 220 nm in size (Figure 1). Further characterization of the SjEVs by transmission electron microscopy revealed EVs to be round vesicles of approximately 100 - 200 nm in diameter (Figure 2). Western blotting analysis indicated that the isolated SjEVs were recognized using CD63 antibodies, which is a typical EV marker (Figure 3). Fluorescence microscopy observation indicated that the PKH67-labeled SjEVs were internalized by the NCTC clone 1,469 cells and that the SjEVs were mainly distributed in the cytoplasm of recipient cells (Figure 4).
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Samples</td>
			<td>EVs/100ml culture medium</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>250 &#181;g</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>208 &#181;g</td>
		</tr>
		<tr>
			<td>#3</td>
			<td>220 &#181;g</td>
		</tr>
	</tbody>
</table>
Table 1: SjEVs protein obtained from culture medium; the amount of SjEVs accessed by BCA assay per 100 mL of culture medium, containing ~500 worm pairs, is shown.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57514/57514fig1.jpg" /> 
Figure 1: Size distribution of SjEVs Isolated from cultured medium. 20 &#181;L of SjEVs solution was diluted with 1000 &#181;L PBS; then, the mixture was analyzed using a high performance two angle particle and molecular size analyzer. The result shown is representative of the data from three independent isolations. <a href="/files/ftp_upload/57514/57514fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57514/57514fig2.jpg" /> 
Figure 2: Characterization of SjEVs by transmission electron microscopy. <a href="/files/ftp_upload/57514/57514fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57514/57514fig3.jpg" /> 
Figure 3: Western blotting analysis of the isolated SjEVs against CD63 antibodies. <a href="/files/ftp_upload/57514/57514fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57514/57514fig4.jpg" /> 
Figure 4: Uptake of SjEVs by mammalian cells. The PKH labeled SjEVs are shown in green, while the cell nuclei are shown in blue. <a href="/files/ftp_upload/57514/57514fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

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Nanyang Technological University

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7.8K Views.  Ministry of Agriculture. Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

Video: Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and mammalian final host, respectively. The ability to isolate and sustain the different life cycle stages under in vitro culture conditions has greatly facilitated investigations of the cellular, biochemical and molecular mechanisms regulating parasite growth, development and host interactions. Transmission of schistosomiasis requires asexual reproduction and development of multiple larval stages within the snail host; from the infective miracidium, through primary and secondary sporocysts, to the final cercarial stage that is infective to humans. In this paper we present a step-by-step protocol for mass hatching and isolation of Schistosoma mansoni miracidia from eggs obtained from livers of infected mice, and their subsequent introduction into in vitro culture. It is anticipated that the detailed protocol will encourage new researchers to engage in and broaden this important field of schistosome research.

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

This article describes a protocol for the large-scale axenic isolation and collection of free-swimming miracidia of the human blood fluke Schistosoma mansoni and their subsequent processing for introduction into in vitro culture.

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and ...

Developmental Biology

An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's interstitial space. The efficient enrichment of these sEVs is an important prerequisite to the study of their biological function and a key to the development of clinical detection techniques and therapeutic carrier technology. It is difficult to isolate sEVs from tissue because they are usually heavily contaminated. This study provides a method for the rapid enrichment of high-quality sEVs from liver cancer tissue. The method involves a four-step process: the incubation of digestive enzymes (collagenase D and DNase &#921;) with tissue, filtration through a 70 &#181;m cell strainer, differential ultracentrifugation, and filtration through a 0.22 &#181;m membrane filter. Owing to the optimization of the differential ultracentrifugation step and the addition of a filtration step, the purity of the sEVs obtained by this method is higher than that achieved by classic differential ultracentrifugation. It provides an important methodology and supporting data for the study of tissue-derived sEVs.

Here we describe the enrichment of small extracellular vesicles derived from liver cancer tissue through an optimized differential ultracentrifugation method.

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's ...

Cancer Research

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

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In particular, small extracellular vesicles (sEVs) act as nano-carriers transferring natural antigens, which are critical in host immunomodulation and parasite survival. This article presents a step-by-step protocol to isolate sEVs from larval stage cultures of Echinococcus granulosus and analyzes their uptake by dendritic cells obtained from murine bone marrow, which acquire adhesion and antigen presentation capacity during their maturation after one week of in vitro culture. This article provides comprehensive information for generating, purifying, and quantifying sEVs using ultracentrifugation alongside parallel analyses of dynamic light scattering and transmission electron microscopy. Additionally, a detailed experimental protocol is outlined for isolating and cultivating mouse bone marrow cells and driving their differentiation into dendritic cells using Flt3L. These dendritic cells can present antigens to na&#239;ve T cells, thereby modulating the type of immune response in vivo. Thus, alternative protocols, including confocal microscopy and flow cytometry analysis, are proposed to check the acquired maturational phenotype of dendritic cells previously exposed to parasitic sEVs. Finally, it is worth noting that the described protocol can be applied as a whole or in individual parts to carry out parasite in vitro culture, isolate extracellular vesicles, generate bone marrow-derived dendritic cell cultures, and perform uptake assays with these cells.

Here, we describe in vitro culture conditions, isolation, and increased generation of extracellular vesicles (EVs) from Echinococcus granulosus. The small EVs were characterized by dynamic light scattering and transmission electron microscopy. The uptake by bone marrow-derived dendritic cells and their phenotypic modulation were studied using confocal microscopy and flow cytometry.

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Biology

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 ...

Medicine

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...

Enhanced Bacterial Extracellular Vesicles Isolation for Microbiota Multi-Omics

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs.
This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg.
Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

Author Spotlight: Accelerating Research on Bacterial Extracellular Vesicles Separation and Heterogeneity 

This study describes a method to isolate and purify bacterial extracellular vesicles (BEVs) enriched from human feces via density gradient centrifugation (DGC), identifies the physical characteristics of BEVs from morphology, particle size, and concentration, and discusses the potential applications of the DGC approach in clinical and scientific research.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

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Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue

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

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation