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 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

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...

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

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 ...

isolation-characterization-extracellular-vesicles-from-adult

Research

JoVE Journal

Immunology and Infection

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

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 ...

Forschung

Immunologie und Infektion

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

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Isolierung und Charakterisierung von dendritischen Zellen und Makrophagen aus dem Mausdarm

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

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.
In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.
A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.
We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

Vereinfachte Menschen Neutrophil Extracellular Traps (NETs) Isolierung und Handhabung

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

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

Isolierung und Charakterisierung von Neutrophilen mit Anti-Tumor-Eigenschaften

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

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Isolierung, Charakterisierung und funktionelle Untersuchung des Zahnfleisch 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

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 microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

Isolierung und Durchflusszytometrie Charakterisierung von murinen Dünndarms Lymphozyten

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

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

Fluss Virometry zur Analyse Antigenic Spektren von Virionen und extracellular Vesikeln

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

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

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

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Legionella pneumophila der äußeren Membran - Vesikeln: Isolierung und Analyse ihrer Pro-inflammatorische Potential auf Makrophagen

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

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Isolierung, Charakterisierung und Reinigung der Makrophagen aus den Geweben von Fettleibigkeit bedingte Entzündung betroffen

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

Isolierung und Charakterisierung von Neutrophilen abgeleitet Mikropartikel für funktionelle Studien

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...