Name: isolation and characterization of extracellular vesicles from adult schistosoma japonicum
Uploaded: 2018-05-22T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum at JoVE.com

Denna studie var, delvis eller i sin helhet, stöds av National Natural Science Foundation Kina (31472187 och 31672550) och The Agricultural Science och Technology Innovation Program av den kinesiska Vetenskapsakademien jordbruket.

Alla djurförsök godkändes av den lokala etiska kommittén för Shanghai veterinärmedicinska Research Institute, kinesiska jordbruket vetenskapsakademin (tillåta nummer: SHVRIAU-14-0101).
1. in Vitro kultur av Schistosomes
Obs: Schistosomes representerar ett biohazard. Arbetstagare bör bära latex handskar på alla tider när hantering maskar, schistosomal suspensioner eller andra relaterade biologiska material. Nya Zee...

<ol>
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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. 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Nyligen genomförda undersökningar om EVs har visat att schistosome EVs spelar en viktig roll i värd-patogen interaktioner3,9,12,16. Till ytterligare adress deras reglerande funktioner, är det viktigt att isolera EVs från schistosomes. Här beskriver vi en alternativ metod för SjEV isolering. Denna metod ger en rad storlek på SjEVs, från 100 nm till 400 nm, i vuxen S. japonicum....

Extracellulära blåsor (EVs) är en population av små membran-bundna blåsor inkapslade med olika proteiner, lipider och nukleinsyror. Senare studier visat att EVs spelar en avgörande roll i cell-cell kommunikation, och är involverade i regleringen av många fysiologiska processer, inklusive cell utveckling, immunreglering, angiogenes och cell migration2, 3,4,5. Ackumulerande bevis indikerar att EVs, cirkulerande exosomes och deras miRNA Last representerar potentiella biomarkörer för vissa sjukdomar6.
Flera protozoer såsom Trichomonas vaginalis, Trypanosoma cruzioch Leishmania spp., har visat sig kunna utsöndra EVs; maskar har dessutom visat att utsöndra EVs till levande värd7. Parasitiska EVs har visat sig vara inblandade i underhållet av infektion, patogenicitet8och immunreglering9. Senaste studier i schistosomes båda Schistosoma mansoni (S. mansoni) 10,11 och S. japonicum12 har uppgett att vuxna schistosomes utsöndrar exosome-liknande blåsor som kan vara involverade i funktionella reglementet av specifika biologiska processer.
Hittills har har flera metoder använts för att isolera extracellulära vesicles, såsom ultracentrifugering, ultrafiltrering13, användning av polymerbaserade reagenser, storlek-utslagning kromatografi och immunoaffinity isolering. Dessa olika metoder har sina egna fördelar och begränsningar14. Allmänhet, ultracentrifugering anses metoden guldmyntfoten för vesikler isolering. Dock lider denna metod begränsning av potentiella EV aggregering14.
I schistosomes, ett par metoder har rapporterats för EV isolering: dessa inkluderar ultracentrifugering11,12,10 och användningen av kommersiella EV isolering kit16 för flera etapper (ägg16, schistosomula11, vuxen schistosomes10,12,17). Med tanke på att microvesicles har ett brett utbud av storlek från flera hundra nanometer till tusen nanometer, utvecklat vi en alternativ metod som kombinerar med användning av bänk centrifug, ultrafiltrering och en kommersiell EV isolering kit att isolera EVs från Adult Schistosoma japonicum. Isolerade EVs vanligtvis besatt en diameter på 100-400 nm och var framgångsrikt internaliseras av mottagarens celler.

<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

Extracellulära blåsor (EVs) är membranös blåsor utgivet en mängd celler i den extracellulära mikromiljö. EVs representerar en befolkning på heterogena vesicles, vars storlekar mellan 40 och 1000 nm. Samlade bevis anges att EVs spelar viktiga reglerande roller i patogen-värd-interaktioner. En djup förståelse av schistosome EVs bör ge insikter om mekanismerna bakom schistosome-värd-interaktioner, möjliggöra utveckling av nya strategier mot snäckfeber. Här, vi strävar efter att ytterligare studera EVs funktioner i schistosomes genom att presentera ett protokoll för isolering och karakterisering av EVs från vuxna Schistosoma japonicum (S. japonicum). EVs isolerades från in vitro- odlingsmedium med centrifugering kombinerad med en kommersiell exosome isolering kit. Den isolerade S. japonicum EVs (SjEVs) normalt har en diameter på 100-400 nm och kännetecknas av överföring elektroniska mikroskopi och western blotting. Användningen av PKH67 dye-märkt SjEVs har visat att SjEVs är internaliserad av mottagarens celler. Sammantaget ger våra protokoll en alternativ metod för att isolera EVs från vuxna schistosomes; den isolerade SjEVs kan vara lämplig för funktionell analys.

För att kvantifiera utbytet av isolerade SjEVs med beskrivna protokollet, brukade vi BCA protein assay åt protein koncentrationen av den isolerade SjEVs från 28-dagars vuxen schistosomes. I tabell 1visas SjEV protein koncentrationen varierade 208 µg 250 µg per 100 mL medium (tabell 1). Partikelstorlek, som bestäms av Malvern nanopartiklar analys, anges att den isolerade SjEVs varierade från 100 nm till 400 nm, med den högsta befolkningen runt 220 ...

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

Isolering och karakterisering av extracellulära blåsor från Adult Schistosoma japonicum

Här presenterar vi ett protokoll för att beskriva extracellulära blåsor (EVs) isolering i in vitro- odlade medium från vuxna Schistosoma japonicum.

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

Research

JoVE Journal

Immunology and Infection

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Cercarial Transformation och In vitro Odling av Schistosoma mansoni Schistosomules

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

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

Använda ägg från<em&gt; Schistosoma mansoni</em&gt; Som en<em&gt; In vivo</em&gt; Modell av inälvsmask-lunginflammation

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

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Isolering och karakterisering av dendritiska celler och makrofager från mus tarm

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

Förenklad Human Neutrofila Extracellulär Fällor (NET) Isolering och Hantering

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Isolering och karakterisering av neutrofiler med Anti-tumöregenskaper

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Isolering, karakterisering och funktionella Undersökning av tandkötts Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

Isolering och Flödescytometriska karakterisering av murina tunntarms lymfocyter

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Flöde Virometry Analysera Antigen Spectra virioner och extracellulär Blåsor

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Legionella pneumophila yttre membranvesiklar: Isolering och analys av deras pro-inflammatoriska Potential på makrofager

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...