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 inf...

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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 ...

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 border="0" cellpadding="0" cellspacing="0" style="width:1161px;" width="1160">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="31">
			<td height="31" style="height:31px;width:459px;">Material</td>
			<td style="width:196px;"></td>
			<td style="width:188px;"></td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:459px;">Total Exosome Isolation Reagent (from cell culture media)</td>
			<td style="width:196px;">ThermoFisher SCIENTIFIC</td>
			<td style="width:188px;">4478359</td>
			<td style="width:319px;">For isolating S. japonicum extracellular vesicles</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:459px;">PKH67</td>
			<td style="width:196px;">Sigma-aldrich</td>
			<td style="width:188px;">MINI67</td>
			<td style="width:319px;">For labeling Evs</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">RPMI 1640 Medium</td>
			<td style="width:196px;">ThermoFisher SCIENTIFIC</td>
			<td>11875119</td>
			<td>For parasite culture</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Penicillin-Streptomycin</td>
			<td style="width:196px;">ThermoFisher SCIENTIFIC</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">0.22&#956;m syringe filter</td>
			<td style="width:196px;">Merck</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">SnakeSkin Dialysis Tubing</td>
			<td style="width:196px;">Thermo SCIENTIFIC</td>
			<td>68035</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">PEG8000</td>
			<td style="width:196px;">Sangon Biotech</td>
			<td>A100159&#160;</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">RIPA</td>
			<td style="width:196px;">Beyotime</td>
			<td>P0013B</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">EMD Millipore&#8482; Immobilon&#8482; Western Chemiluminescent HRP Substrate (ECL)</td>
			<td style="width:196px;">Fisher scientific</td>
			<td>WBKLS0100&#160;</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">DAPI</td>
			<td>Cell Signaling Technology</td>
			<td>4083</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">BCA kit</td>
			<td style="width:196px;">Beyotime</td>
			<td>P0010</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">CD63 antibodies</td>
			<td style="width:196px;">Sangon Biotech</td>
			<td>D160973-0025</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">PVDF</td>
			<td>Bio-Rad</td>
			<td>1704273</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Formvar/Carbon 400 mesh</td>
			<td>Ted Pella</td>
			<td>01754-F</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Phosphotungstic Acid</td>
			<td>Ted Pella</td>
			<td>19402</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;">anti-mouse IgG-HRP</td>
			<td>CWBIO</td>
			<td>CW0102</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:459px;">NCTC clone 1469 cells</td>
			<td style="width:196px;">ATCC</td>
			<td style="width:188px;">ATCC&#174; CCL-9.1&#8482;</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:459px;">FBS</td>
			<td style="width:196px;">HyClone</td>
			<td style="width:188px;">SV30087.02</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:459px;">Equipments</td>
			<td style="width:196px;"></td>
			<td style="width:188px;"></td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">GE chemoluminescance imaging system</td>
			<td>GE</td>
			<td>ImageQuant LAS4000mini</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">Transmission electron microscopy</td>
			<td>Hitachi</td>
			<td>H-7600</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Milli-Q water</td>
			<td>Milli-Q</td>
			<td>Milli-Q Elix</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Eppendor Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5804R</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">Zetasizer Nano</td>
			<td>Malvern</td>
			<td>Zetasizer Nano ZS&#160;</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Ultracentrifuge</td>
			<td style="width:196px;">Beckman</td>
			<td colspan="2">Optima L-100 XP Ultracentrifuge</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Incubator</td>
			<td style="width:196px;">ESCO</td>
			<td>CCL-107B</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Microscope</td>
			<td style="width:196px;">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 pop...

Watch this Scientific Journal Video about Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum at JoVE.com

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 ...

The overall goal of this procedure is to isolate extracellular vesicles from the medium of in vitro cultured adult Schistosoma japonicum. This method can efficiently isolate EV in in vitro culture medium from adult Schistosoma japonicum. The main advantage of this technique is able to efficiently isolate extracellular vesicle from short-term in vitro culture medium, which can minimize the stress potentially result in non-physiological EV secretion.Demonstration will be by Juntao Liu, a technician from my lab. Collect the parasites in a 500-milliliter beaker, and wash them thoroughly but gently three times using 200 milliliters of warm PBS. Keep the washes brief, and use gravity sedimentation to separate the parasites.Next, onto 90-millimeter dishes, plate about 200 worm pairs per dish. In the dish, wash the parasites twice more using gravity sedimentation. For these washes, use warm RPMI medium supplemented with pen-strep.Replace the second wash with 40 milliliters of the medium without antibiotics. Then, culture the parasites at 37 degrees Celsius under 5%carbon dioxide for two hours. After two hours, transfer the media from the plates to centrifuge tubes, and then pool the eggs and worm debris at 2, 000 g for 30 minutes at four degrees Celsius.Then, collect and transfer the supernatant to a fresh tube. Next, filter the supernatant through a 0.22-micron syringe filter, and collect the filtered solution into a dialyzing bag with a molecular weight cut-off of 3.5 kilodaltons. Then, dialyze the solution using 8%PEG8000 solution at four degrees Celsius overnight.This step is important for increasing the yield of extracellular vesicles. The next day, transfer the dialyzed solution to a new tube, and add 0.5 volumes of the total exosome isolation reagent. Mix the solution until it is homogeneous, and incubate at about five degrees Celsius overnight.The following morning, centrifuge the mixture at 15, 000 g for one hour at about five degrees Celsius. Next, aspirate and discard the supernatant, and resuspend the pellet of EVs in two milliliters of PBS. The pellet will not likely be visible.For western blot analysis, first determine the protein concentration of the sample, such as with the BCA assay. Then, prepare 10 to 20 micrograms of EVs in 22.5 microliters of RIPA buffer. Next, add 7.5 microliters of 4X loading buffer, and heat the mixture at 95 degrees Celsius for five minutes.After the heat treatment, separate the extracellular vesicle proteins on a 12%SDS-PAGE gel. After running the gel, the proteins are transferred into PVDF membrane. Probe the membrane with anti-CD63 antibodies at a one to 1, 000 dilution.Then, treat the membrane with anti-rabbit IgG-HRP at a one to 5, 000 dilution. Now, use an ECL detection reagent to develop the membrane, and follow with analysis on a chemiluminescence imaging system. Start with transferring five micrograms of sample into a polypropylene ultracentrifuge tube, and adjust the volume to 12 milliliters with RPMI-1640 medium.Then, centrifuge the sample at 110, 000 g for 90 minutes at four degrees Celsius. After the centrifugation, carefully aspirate and discard the supernatant. Then, prepare a 2X extracellular vesicle suspension by adding one milliliter of diluent C to the pellet.Resuspend the pellet with gentle pipetting. Now, prepare fresh 2X dye solution. Add two microliters of the PKH67 ethanolic dye to one milliliter of diluent C in a microfuge tube, and mix well.Then, stain the sample by mixing together the two solutions and incubating the mix at room temperature for four minutes. Next, stop the staining by adding four milliliters of FBS and waiting another minute. Then, add RPMI-1640 medium to adjust the volume to 12 milliliters, and spin down the suspension to isolate the stained vesicles.Remove and discard the supernatant. Then, resuspend the pelleted PKH67-labeled EVs in 12 milliliters of RPMI-1640 medium. Then, repeat the centrifugation, but this time collect the vesicles in one milliliter of PBS.For the uptake assay, prepare overnight cultures of mammalian cells in six-well plates. The next morning, add five micrograms of PKH67-labeled vesicles to each well, and continue the culture for two to four hours. After the incubation, remove the medium, and wash the cells with PBS two times.Then, fix the cells for 15 minutes, wash them twice with PBS, and stain them with DAPI before imaging. Schistosomes at the liver stage were collected from rabbits 28 days post-infection. Extracellular vesicles were then collected from the schistosomes worms and analyzed.Their particle size, as determined by Malvern nanoparticle analysis, ranged between 100 and 400 nanometers, with the bulk of the population near 220 nanometers. Transmission electron microscopy revealed the extracellular vesicles to be round and between 100 and 200 nanometers in diameter. Western blotting analysis indicated that the isolated extracellular vesicles were recognized using CD63 antibodies, which is a typical extracellular vesicle marker.Fluorescence microscopy of PKH67-labeled extracellular vesicles internalized by the NCTC clone 1469 cells showed that the extracellular vesicles were mainly distributed in the cytoplasm of recipient cells. After watching this video, you should have a good understanding of how to isolate EV in in vitro culture medium from adult Schistosoma japonicum.

isolation-characterization-extracellular-vesicles-from-adult

Research

JoVE Journal

Immunology and Infection

7.8K Görüntüleme.  Ministry of Agriculture. The overall goal of this procedure is to isolate extracellular vesicles from the medium of in vitro cultured adult Schistosoma japonicum. This method can efficiently isolate EV in in vitro culture medium from adult Schistosoma japonicum. The main advantage of this technique is able to efficiently isolate extracellular vesicle from short-term in vitro culture medium, which can minimize the stress potentially result in non-physiological EV secretion.Demonstration will be by Juntao Liu, a...