Name: isolation and characterization of extracellular vesicles from adult schistosoma japonicum
Uploaded: 2018-05-22T13:00:00.000+00:00
Duration: 7 min 37 s
Description: Watch this Scientific Journal Video about Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum at JoVE.com

Diese Studie war, im Teil oder im ganzen, unterstützt durch die National Natural Science Foundation of China (31472187 und 31672550) und The Agricultural Science and Technology Innovation Program von der chinesischen Akademie der Agrarwissenschaften.

Alle Tierversuche wurden genehmigt durch die lokale Ethikkommission des Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (erlauben Nummer: SHVRIAU-14-0101).
1. in-vitro- Kultur von Schistosomen
Hinweis: Schistosomen repräsentieren ein Biohazard. Arbeiter tragen Latex Handschuhe überhaupt mal beim Umgang mit Würmern, schistosomal Suspensionen oder anderen biologische Materialien im Zusammenhang. Neuseeland-Kaninchen wurden mit ~ 2.000 Cercariae über Abdominal-Belichtung infiziert. Schistosomen Stadium der Leber wurden gesammelt von Kaninchen mit S. Japonicum Cercariae auf 28 Tage nach der Infektion durch Durchblutung der Leber und mesenterialen Adern angesteckt. Die Verfahren der Wurm Sammlung haben in früheren Publikationen Berichten Studien an Mäusen18verwiesen.
<ol><li>Parasiten in einen 500-mL-Becherglas zu sammeln. Waschen Sie sie gründlich und sanft 3 X mit 200 mL vorgewärmt (37 ° C) PBS (pH 7,4).</li>
	<li>~ 200 Wurm Paare für jedes 90 mm Petrischale zu trennen. Waschen Sie die Parasiten gründlich und sanft 2 X mit 50 mL vorgewärmt (37 ° C) RPMI-1640 Kulturmedium mit 100 U von Penicillin und 100 mg/mL von Streptomycin.</li>
	<li>Pflegen Sie Parasiten in 40 mL vorgewärmten RPMI-1640 Kulturmedium ohne Antibiotika bei 37 ° C weniger als 5 % CO2 bei einer Dichte von ~ 5 Wurm Paare /mL für 2 Stunden in 90 mm Petri dish. Noten: RPMI 1640 Medium sollte nicht Serum enthalten, ansonsten exogene extrazelluläre Vesikel vorgestellt werden.</li>
</ol>(2) vorbehandelten Zustand Medien
<ol><li>Sammeln Sie das Kulturmedium zu und Zentrifugieren Sie das Medium bei 4 ° C bei 2.000 × g für 30 min Eiern entfernen und Wurm tegmental Schutt.</li>
	<li>Übertragen Sie den überstand auf eine frische Rohr und Filtern Sie dann die Lösung mit Hilfe eines Filters 0,22 µm Spritze.</li>
	<li>Die filtrierte Lösung in einem dialysierter Beutel zu sammeln (Molekulargewicht Cut-Off: 3.5kD) und dann die Lösung mit 8 % PEG8000 Lösung bei 4 ° C über Nacht Dialyse. Noten: Dieser Schritt ist wichtig, SjEVs zu bereichern, zur Erhöhung der Ausbeute der SjEVs Isolation.</li>
</ol>(3) die Isolierung des SjEVs
<ol><li>Übertragen der dialysierten Lösung zu einem neuen Schlauch und fügen Sie 0,5 Volumen des gesamten exosom Isolierung Reagens.</li>
	<li>Mischen Sie die Lösung gut durch aufschütteln oder pipettieren rauf und runter um die Homogenität der Lösung zu gewährleisten.</li>
	<li>Inkubieren Sie die Mischung bei 2 ° C bis 8 ° C über Nacht.</li>
	<li>Zentrifugieren Sie die Mischung bei 15.000 × g für 1 h bei 2 ° C bis 8 ° C.</li>
	<li>Aspirieren und den überstand verwerfen. Noten: EVs enthalten die Kugel am unteren Ende der Röhre (in den meisten Fällen nicht sichtbar).</li>
	<li>Aufschwemmen der EVs Pellet in 2 mL PBS und speichern Sie anschließend die EVs-Lösung bei-80 ° C bis zur weiteren Analyse.</li>
</ol>4. Charakterisierung der EVs von Western Blotting
<ol><li>Bestimmen Sie die Konzentration des Proteins der SjEVs Probe, z. B. durch BCA-Assay.</li>
	<li>10-20 µg des EFD in 22,5 µL RIPA Puffer vorzubereiten. Dann fügen Sie 7,5 µL 4 x laden Puffer und Hitze für 5 min bei 95 ° C.</li>
	<li>Trennen Sie SjEVs Proteine durch 12 % SDS-PAGE Gel.</li>
	<li>Die Proteine auf PVDF-Membran übertragen und dann mit Anti-CD63-Antikörper (1:1, 000 Verdünnung), gefolgt von sondieren mit Anti-Kaninchen IgG-HRP (1:5, 000 Verdünnung) Sonde.</li>
	<li>Verwenden Sie ECL-Erkennung Reagenz für Membranentwicklung und erkennen Sie die Signale zu, indem eine Chemilumineszenz imaging-System.</li>
</ol>5. Charakterisierung der EVs von Transmissions-Elektronenmikroskopie
<ol><li>200 Mesh Formvar-beschichtete Netze 2 µL der isolierten SjEVs hinzu und dann bei Raumtemperatur trocknen lassen.</li>
	<li>Färben mit 2 % Phosphotungstic Acid für 1 min und dann bei Raumtemperatur trocknen lassen.</li>
	<li>Laden Sie die Gitter auf dem Probenhalter des Transmissions-Elektronenmikroskop und einen 80 kV Elektronenstrahl für Bilderfassung ausgesetzt.</li>
</ol>6. SjEVs Label mit PKH67 und Zelle-Aufnahme-Test
Noten: Alle weiteren Schritte wurden bei Raumtemperatur (20-25 ° C), durchgeführt, sofern nicht anders angegeben.
<ol><li>5 µg SjEVs Lösung in einem konischen Boden aus Polypropylen Röhrchen zu übertragen und die Lautstärke bis 12 mL durch Hinzufügen von RPMI 1640 Medium.</li>
	<li>Zentrifugieren Sie die SjEVs bei 110.000 × g für 90 min bei 4 ° C um SjEVs pellet.</li>
	<li>Aspirieren Sie nach Zentrifugation sorgfältig überstand. Hinweis: Der PKH67 ethanolische Farbstoff sollte nicht direkt an das Pellet SjEV hinzugefügt werden, heterogene Färbung und reduzierte Zellviabilität sonst würde.</li>
	<li>Bereiten Sie eine 2 X EV Suspension Lösung durch Zugabe von 1 mL Verdünnungsmittel C zum Pellet SjEV; um komplette Dispersion zu gewährleisten, mit sanften pipettieren aufzuwirbeln.</li>
	<li>Kurz vor der Färbung im Verdünnungsmittel C, bereiten Sie einen frischen 2 x Farbstofflösung durch 1 mL Verdünnungsmittel C in einem Polypropylen Zentrifugenröhrchen 2 µL der PKH67 ethanolische Farbstofflösung hinzufügen und mischen um gut zu zerstreuen.</li>
	<li>Fügen Sie schnell die 1 mL 2 X EV Suspension (Schritt 6.4) zu 1 mL 2 x Farbstofflösung (Schritt 6.5) und sofort mischen die Probe durch pipettieren.</li>
	<li>Inkubieren Sie die Mischung bei Raumtemperatur für 4 min.</li>
	<li>Die Färbung durch Zugabe von 4 mL FBS zu stoppen und dann 1 min inkubieren.</li>
	<li>Fügen Sie RPMI1640 Medium, um die Lautstärke zu 12 mL und dann bei 110.000 × g bei 4 ° C für 90 min zentrifugieren.</li>
	<li>Entfernen des Überstands und RPMI1640 Medium, um die PKH67 mit der Bezeichnung SjEV Pellets aufzuwirbeln.</li>
	<li>Zentrifugieren Sie bei 110.000 × g bei 4 ° C für 90 min. einmal waschen und dann hinzufügen PBS um die SjEVs Pellets aufzuwirbeln.</li>
</ol>7. SjEVs Zelle Aufnahme Assay
<ol><li>Samen Säugerzellen wie NCTC Klonen 1469 Zellen oder HEK293T in 6-Well-Platten (2 × 105 Zellen pro Well) und über Nacht Kulturzellen.</li>
	<li>5 µg PKH67 versehenen SjEVs hinzufügen und dann Kulturzellen bei 37 ° C für 2-4 h.</li>
	<li>Entfernen Sie nach der Inkubation das Medium und waschen Sie die Zellen mit PBS zweimal.</li>
	<li>Befestigen Sie die Zellen mit 4 % Formaldehyd-Lösung für 15 min und zweimal mit PBS gewaschen.</li>
	<li>Fleck Zellkerne mit 4 ', 6-Diamidino-2-Phenylindole (DAPI).</li>
	<li>Beobachten Sie die Zellen mittels Fluoreszenzmikroskopie.</li>
</ol>

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Die Autoren haben nichts preisgeben.

Neuere Studien über EFD nachgewiesen, dass Schistosome EVs spielen eine wichtige Rolle in Wirt-Pathogen Interaktionen3,9,12,16. Weitere Adresse unbedingt ihre Regulierungsaufgaben EVs von Schistosomen zu isolieren. Hier beschreiben wir eine alternative Methode zur SjEV Isolierung. Diese Methode führt zu eine breiten Palette Größe von SjEVs, von 100 nm bis 400 nm, bei Erwachsenen S. Japo...

Extrazelluläre Vesikel (EVs) sind eine Bevölkerung von kleinen Membrane-springen Vesikel mit verschiedenen Proteine, Lipide und Nukleinsäuren gekapselt. Jüngste Studien gezeigt, dass EVs eine entscheidende Rolle in der Zell-Zell-Kommunikation spielen und sind beteiligt an der Regulation des zahlreiche physiologische Prozesse, einschließlich der Zellentwicklung, Immunregulation, Angiogenese und Zelle Migration2, 3,4,5. Sammeln Beweise gibt, dass EVs, zirkulierenden exosomen und ihre Fracht MiRNA potenzielle Biomarker für bestimmte Krankheiten6darstellt.
Einige Protozoen wie Trichomonas Vaginalis, Trypanosomen Trypanosomaund Leishmania Spp., haben gezeigt, dass EVs; absondern zu können Helminthen zusätzlich gefunden wurden, EVs in lebenden absondern beherbergt7. Parasitäre EVs wurden gezeigt, bei der Aufrechterhaltung der Infektion, Pathogenität8und Immunregulation9einbezogen werden. Aktuelle Studien in Schistosomen beide Schistosoma Mansoni (S. Mansoni) 10,11 und S. Japonicum12 haben gezeigt, dass Erwachsene Schistosomen exosom erinnernden Bläschen absondern, die möglicherweise die funktionale Vorschriften bestimmte biologische Prozesse beteiligt.
Bisher wurden verschiedene Methoden zur extrazellulären Vesikel, wie Ultrazentrifugation, Ultrafiltration13, die Verwendung von Polymer-basierten Reagenzien, Größe-Ausschluss-Chromatographie und immunoaffinitäts Isolation isolieren. Diese verschiedenen Methoden besitzen ihre eigenen Vorzüge und Grenzen14. Im Allgemeinen gilt Ultrazentrifugation die Goldstandard-Methode für die Vesikel Isolierung. Allerdings leidet diese Methode aus der Beschränkung des potenziellen EV Aggregation14.
In Schistosomen, eine Reihe von Methoden zur Isolierung der EV gemeldet wurden: dazu gehören Ultrazentrifugation11,12,10 und die Verwendung von kommerziellen EV Isolation Kit16 für mehrere Stufen (Eiern16, Schistosomula11, Erwachsene Schistosomen10,12,17). Da die Microvesicles einer breiten Palette von mehreren hundert Nanometer bis tausend Nanometer groß sind, entwickelten wir einer alternativen Methode, die kombiniert mit dem Einsatz von Bank-Zentrifuge, Ultrafiltration und eine kommerzielle EV isolierungskit der EVs von isolieren Erwachsenen Schistosoma Japonicum. Die isolierten EVs in der Regel besaß einen Durchmesser von 100-400 nm und wurden erfolgreich durch den empfängerzellen verinnerlicht.

<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

Extrazelluläre Vesikel (EVs) sind häutig Vesikel, die von einer Vielzahl von Zellen in der extrazellulären Mikroumgebung veröffentlicht. EFD repräsentieren eine Bevölkerung von heterogenen Vesikel, deren Größe zwischen 40 und 1.000 nm. Gesammelte Beweise gezeigt, dass EVs in Erreger-Wirt Interaktionen eine wichtige regulatorische Rolle spielen. Ein tiefes Verständnis der schistosome-EVs soll Einblicke in die Mechanismen schistosome--Wirt Interaktionen, ermöglicht die Entwicklung neuer Strategien gegen Bilharziose. Hier wollen wir weiter studieren EVs Funktionen in Schistosomen durch Vorlage eines Protokolls für die Isolierung und Charakterisierung des EFD vom Erwachsenen Schistosoma Japonicum (S. Japonicum). EVs wurden von in-vitro- Kulturmedium mit Zentrifugation kombiniert mit einem kommerziellen exosom isolierungskit isoliert. Die isolierten S. Japonicum EVs (SjEVs) in der Regel besitzen einen Durchmesser von 100-400 nm, und zeichnen sich durch Übertragung elektronischer Mikroskopie und westliche Beflecken. Die Verwendung von PKH67 Farbstoff-markierten SjEVs hat gezeigt, dass SjEVs durch den empfängerzellen internalisiert werden. Insgesamt bietet unser Protokoll eine alternative Methode zur Isolierung von EVs von Erwachsenen Schistosomen; die isolierten SjEVs möglicherweise für Funktionsanalyse geeignet.

Um die Ausbeute an isolierten SjEVs mit Hilfe der beschriebenen Protokoll zu quantifizieren, haben wir die Konzentration des Proteins von der isolierten SjEVs von 28 Tagen Erwachsene Schistosomen Zugriff auf BCA Protein Assay verwendet. Wie aus Tabelle 1hervorgeht, reichte die SjEV Proteinkonzentration von 208 µg bis 250 µg pro 100 mL Medium (Tabelle 1). Die Partikelgröße Malvern Nanopartikel Analyse bestimmt darauf hingewiesen, dass die isolierte SjE...

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Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Isolierung und Charakterisierung von extrazellulären Vesikel von Erwachsenen Schistosoma japonicum

Hier präsentieren wir Ihnen ein Protokoll zur extrazellulären Vesikel (EVs) Isolierung in in Vitro kultivierten Medium von Erwachsenen Schistosoma Japonicumbeschreiben.

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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Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

7.8K Aufrufe.  Ministry of Agriculture. Hier präsentieren wir Ihnen ein Protokoll zur extrazellulären Vesikel (EVs) Isolierung in in Vitro kultivierten Medium von Erwachsenen Schistosoma Japonicumbeschreiben.

Serie: 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.

Zerkarien Transformation und In-vitro- Anbau von Schistosoma mansoni Schistosomules

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

Forschung

Immunologie und Infektion

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.

Mit den Eizellen<em&gt; Schistosoma mansoni</em&gt; Als ein<em&gt; In vivo</em&gt; Modell von Helminthen-induzierten Lungenentzündung

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.

Masse isoliert und In-vitro- Kultivierung von Intramolluscan Stadien der menschlichen Blut 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 ...

Entwicklungsbiologie

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.

Eine Anreicherungsmethode für kleine extrazelluläre Vesikel aus Leberkrebsgewebe

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

Krebsforschung

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.

Größenausschlusschromatographie zur Trennung extrazellulärer Vesikel aus konditionierten Zellkulturmedien

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

Neurowissenschaften

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.

Isolierung und dendritische Zellaufnahme von kleinen extrazellulären Vesikeln aus 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 ...

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.

Reinigung und Analyse von Caenorhabditis elegans Extrazelluläre Vesikel

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

Biologie

<meta charset="utf8"></head><body>Verfeinerungstechniken für die Isolierung von murinen Milz-abgeleiteten Exosomen

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.

<meta charset="utf8"></head><body>Isolierung und Charakterisierung von Exosomen aus Milzgewebe der Maus

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

Isolierung und Charakterisierung von Exosomen aus Milzgewebe der Maus

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

Isolierung und Charakterisierung von extrazellulären Vesikel von Erwachsenen Schistosoma japonicum

Zusammenfassung

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Zerkarien Transformation und In-vitro- Anbau von Schistosoma mansoni Schistosomules

Mit den Eizellen

Masse isoliert und In-vitro- Kultivierung von Intramolluscan Stadien der menschlichen Blut Fluke Schistosoma Mansoni

Eine Anreicherungsmethode für kleine extrazelluläre Vesikel aus Leberkrebsgewebe

Größenausschlusschromatographie zur Trennung extrazellulärer Vesikel aus konditionierten Zellkulturmedien

Isolierung und dendritische Zellaufnahme von kleinen extrazellulären Vesikeln aus Echinococcus granulosus

Reinigung und Analyse von Caenorhabditis elegans Extrazelluläre Vesikel

Isolierung und Charakterisierung von Exosomen aus Milzgewebe der Maus

Isolierung und Charakterisierung von Exosomen aus Milzgewebe der Maus

Isolierung und Charakterisierung von Exosomen aus Milzgewebe der Maus