Die Autoren danken Dr. Le Trinh und Dr. Tobin für die großzügige Bereitstellung der α-Catenin: Citrin Angelschnur und Bill Jackman für die Erlaubnis, die Dreharbeiten in seinem Labor zu tun. Die Autoren erkennen die Finanzierungsquellen National Institutes of Health (Grants 5P20RR016463, 8P20GM103423 und R15AI094406) und USDA (Projekt # ME0-H-1-00517-13). Dieses Manuskript ist als Haupt Land- und Forstwirtschaft Experiment Station Veröffentlichungsnummer 3371 veröffentlicht.

HINWEIS: Alle Zebrafisch Pflegeprotokolle und die Experimente wurden in Übereinstimmung mit NIH-Richtlinien unter Institutional Animal Care und Verwenden Committee (IACUC) Protokoll A2012-11-03 geführt. 1. Zebrafisch Aufzucht bis 4 Tage nach der Befruchtung <ol><li> Sammeln AB Zebrafisch oder andere transgene Linien, innerhalb der ersten 3 Stunden nach der Befruchtung, wie in einem anderen Video 34 gezeigt. </li><li> Inkubieren 120 Eier in eine 15 cm Petrischalen mit 150 ml E3 Medien (5 mM NaCl; 0,17 mM KCl; 0,33 mM CaCl2; 0,33 mM MgCl 2, 2 mM HEPES; pH 6,8) mit Methylenblau (0,3 ug / ml final Konzentration) in einem 33 ° C Inkubator mit 12/12 Hell / Dunkel-Photoperiode. </li><li> Ersetzen Sie die Medien nach 6 Stunden mit E3 + PTU (1-Phenyl-2-thioharnstoff, 10 ug / ml Endkonzentration) und entfernen Sie die toten Eiern. </li><li> Inkubieren für 4 Tage bei 33 ° C, die Medien mit frischen E3 + PTU Austausch nach 2 Tagen. </li></ol> 2.Projektions Micro-Nadel Vorbereitung <ol><li> Ziehen Borosilikat Kapillare (OD 1,2 mm; 0,69 mm ID) in eine Mikronadel mit einem Nadelzieher mit den folgenden Einstellungen (550 Wärme; 0 Pull; 130 Velocity; 110 Time). HINWEIS: Die Einstellungen werden vom Filament variieren Filament ähnlich geformte Mikronadeln zu erhalten (siehe Abbildung 1) und muss neu eingestellt werden, wenn ein neues Filament eingebaut ist. </li><li> Füllen einer Mikronadel mit 3 &amp; mgr; l von 0,4 &amp; mgr; m-filtriert Phosphatpuffersalzlösung (PBS; 137 mM NaCl; 2,7 mM KCl; 10 mM Na 2 HPO 4; 1,8 mM KH 2 PO 4, pH 7,4) unter Verwendung einer 20 ul-Pipette Microloader Tipp. </li><li> Richten Sie das Mikro-Nadel auf einer Mikropipette Halter auf einem Mikromanipulator mit einem Druckinjektionssystem befestigt. Stellen Sie den Einspritzdruck auf 30 PSI und der Impulsdauer bei 30 ms. </li><li> Mit einer Petrischale mit destilliertem Wasser, senken Sie die Mikronadel, bis 1/5 der Nadel in das Wasser. Clip der Mikronadel with feinen Pinzette gerade unter dem Wasserspiegel. Bringen Sie die Mikronadel aus dem Wasser, und drücken Sie das Pedal mehrmals, bis ein Bolus erscheint. </li><li> Den Durchmesser des Bolus durch Absenken der Mikronadel auf einem Hämocytometer mit einem Tropfen des Typs A Sionsöl geschichtet. Stellen Sie die Impulsdauer, um die gewünschte Lautstärke zu erhalten (siehe Tabelle 1). </li><li> Stellen Sie den Gegendruck für den Bolus in Öl stabil bleiben (senken Sie den Druck, wenn der Bolus-Volumen erhöht und erhöht den Gegendruck, wenn der Bolus-Volumen nimmt ab). Notieren Sie sich die Pulsdauer für jede Mikronadel. </li><li> Entfernen Sie das PBS aus der Mikronadel durch Absaugen es mit Hilfe eines Microloader Pipettenspitze und vertreiben die übrig gebliebenen PBS, indem Sie es wieder auf dem Injektor und spiegeln die kontinuierliche Impulsschalter. Reservieren Sie jede Mikronadel. </li></ol> 3. Candida Vorbereitung <ol><li> Streak Candida albicans mit codonoptimierten dTomato, GFP, BFP, EOS oder Far verwandeltRot aus einem gefrorenen Lager (-80 ° C lagerfähig in 25% Glycerin) mit einer sterilen Holzstab auf eine Hefe peptine Dextrose (YPD) Agar-Platte (10 g / l Hefeextrakt, 20 g / l Pepton, 20 g / l Dextrose , 20 g / l Agar, autoklaviert und goss 25 ml in Petrischalen) und Inkubation über Nacht bei 30 ° C. </li><li> Wählen Sie eine Kolonie mit einer sterilen Holzdübel und impfen zu 5 ml YPD-Flüssigkeit (10 g / l Hefeextrakt, 20 g / l Pepton, 20 g / l Dextrose, autoklaviert und aseptisch in 5 ml in 16 x 150 mm Glaskultur aliquotiert Röhren). </li><li> Inkubieren über Nacht bei 30 ° C in einer Rollentrommel bei 60 Upm. </li><li> Sammeln Sie 1 ml C albicans-Kultur und den Transfer in ein 1,7 ml steriles Zentrifugenröhrchen. </li><li> Zentrifugieren bei 5.000 xg für ein paar Sekunden. Leeren Sie den Überstand und 1 ml sterilem PBS, gründlich vortexen. Wiederholen Sie dies zweimal. </li><li> Zählen Sie die Kolonien mit einer Zählkammer durch Verdünnung der 1 ml Lager 1: 1000 in sterilem PBS. </li></ol> 4. Einspritzen in Zebrafischdie Schwimmblase <ol><li> Erwärmen eines Injektionsform (2% Agarose) für 30 min in einem 33 ° C Inkubator. </li><li> Zum gewünschten Zeitpunkt, zu entfernen 100 der 150 ml E3 + PTU von der 15 cm Petrischale die Fische, die zum Einspritzen mit einer 25 ml Pipette ohne Absaugen keine Zebrafisch. </li><li> Anesthetize 4 dpf Zebrafisch durch Zugabe von 2 ml einer 4 mg / ml gepufferte Tricaine Methansulfonat Lager (TR), um die verbleibenden die 50 ml Medium (Endkonzentration 200 ug / ml), und warten Sie 15 Minuten. </li><li> Unter einem Binokular, wählen Sie Fisch mit einem aufgeblasenen Schwimmblase. Fisch kann zu diesem Zeitpunkt auch für homogene Phänotyp gescreent werden, wie beispielsweise neutrophile Verteilung in MPO: GFP-Leitung mit Epifluoreszenz Präpariermikroskop. </li><li> Verdünnen Sie die C albicans Lager aus seiner ursprünglichen Konzentration (Schritt 3.8) in 1,5 x 10 7 koloniebildenden Einheiten pro ml (cfu / ml) in PBS. </li><li> Übertragen Sie 30 Zebrafisch auf die Injektions Gericht mit einem Kunststoffüberführungsrohrtte und entfernen Sie das überschüssige Medien. </li><li> Mit einem Fischerdraht-Werkzeug (in ein Borosilikatglas Kapillaren 0,012 Zoll Durchmesser Angelschnur super geklebt), stellen Sie 3 Zeilen 10 Fische, indem Sie die Injektions Teller vertikal und orientieren die Fische vertikal. </li><li> Entfernen Sie die überzähligen Medien. </li><li> Das Röhrchen gründlich vortexen mit 1,5 × 10 7 cfu / ml C. albicans. </li><li> Füllen einer Mikronadel mit 3 ul C. albicans mit einem neuen Microloader Pipettenspitze und stellen Sie die Pulsdauer auf die entsprechende Einstellung (siehe Schritt 2.9). </li><li> Drehen Sie den Einspritz Gericht, um einen Winkel von 20 ° zwischen der Mikro-Nadel und der Kopf des Fisches zu machen, mit dem Ziel in Richtung der Rückseite des Schwimmblase (3A). </li><li> Drücken Sie den Mikro-Nadel in die Schwimmblase, um sicherzustellen, die Bohrung der Nadel in das Lumen, und drücken Sie das Pedal einmal. </li><li> Wiederholen Sie für jeden Fisch und übertragen zu einer Erholung Schale (25 ml E3 + PTU) durch Fluten der Schale mit einem E3d Entleerung in die stabile Schale. </li><li> Übertragen Sie weitere 30 Fische an der Einspritz Gericht. </li><li> Wiederholen Sie die Injektion mit einer neuen Mikro-Nadel mit gut gefüllten verwirbelt C. albicans. </li><li> Fertig durch Einspritzen der PBS-Kontrolle, wenn nötig, mit einem anderen Gericht Injektion zur Vermeidung von Verunreinigungen und Transfer zu einem separaten Recovery Gericht (Schritt 4.10). </li></ol> 5. Screening der Fisch zum Wunsch Inokulum <ol><li> Vorbereitung 40 ml 0,4% niedrigschmelz Agaroselösung (LMA) durch Zusatz von 160 mg des niedrigen Schmelz Agarose zu 40 ml E3, siedet durch Mikrowellenbehandlung bis zur Auflösung und Abkühlen auf 37 ° C. Fügen Sie 400 ul TR (Endkonzentration 200 ug / ml) nach dem Abkühlen. </li><li> Nach 30 Minuten Erholung, betäuben die Fische, wie beschrieben (Schritt 4.3 mit nur 1 ml TR in 25 ml E3 + PTU, Endkonzentration 200 ug / ml). </li><li> Übertragungs 30 Fisch (mit möglichst wenig Medien wie möglich), um 2 ml LMA in einem Gewichts Boot. </li><li> Saugen Sie einzelne Fische mit einer Transferpipette und Platte den Fisch mit 1 Tropfen LMA in einen 96-Loch-Bildplatte, nur mit den 10 x 6 zentralen Brunnen. </li><li> Wiederholen, bis alle Fische zu Bildschirm werden der Bildplatte geladen. </li><li> Lassen Sie das Medium erstarren bei Raumtemperatur für 5 min. </li><li> Positionieren Sie den Fisch auf ihrer Seite, dafür, dass sie in Kontakt mit dem Glasboden gibt. </li><li> Mit dem 20x-Objektiv auf einem umgekehrten Epifluoreszenz / Konfokalmikroskop und der entsprechenden Filter, notieren Sie die Anzahl der C albicans Hefe-Zellen in jeder Fischschwimmblase. </li><li> Wählen Sie den Fisch mit 15-25 Hefezellen in der Schwimmblase (oder gewünschte Inokulum) euthanize nicht ausgewählte Fisch (800 ug / ml TR in E3) und zurück gewählt Fische einer tiefen Petrischale mit 50 ml E3 + PTU durch Zugabe von 3 Tropfen E3 auf die Bildplatte ausgewählt Brunnen und Absaugen der Fisch mit einer Plastiktransferpipette. </li><li> Inkubiere bei 33 ° C für die gewünschte Dauer. </li></ol> 6. Visualisierung der Fisch-Post-Screening <ol><li> Zu der gewünschten Zeit, entfernen Sie 25 der 50 ml E3 + PTU Medien aus dem tiefen Petrischale die Fische Bild enthält. </li><li> Betäuben des Fisches wie beschrieben (Schritt 4.3 mit nur 1 ml TR in 25 ml E3 + PTU verbleibenden, Endkonzentration 200 ug / ml). </li><li> Platte der Fische in eine 96-Loch-Speicherfolie und Position entsprechend (Schritte von 5,3 bis 5,7). </li><li> Bild durch Scannen der konfokalen Mikroskopie mit den entsprechenden Laser und Emissionsfilter. Zuerst auf den Fisch im 4x Ziel unter differentiellem Interferenzkontrast (DIC) und Bilder der interessierenden Region unter Verwendung der 20X-Objektiv mit der konfokalen Laser im Scanning-Modus mit 2 &amp; mgr; s / Pixel Geschwindigkeit und 1024 x 600 Pixel-Seitenverhältnis zu erwerben. Erwerben Sie Z-Stapel mit 1 &amp; mgr; m-Schrittweite, die Anwendung Kalman-Filter-Modus von 3 Bildern. </li><li> Die Rückkehr zu einem tiefen Petrischale mit 50 ml E3 + PTU oder in individual Vertiefungen (24-well-Kulturschale, 3 ml E3 + PTU) und Inkubation bei 33 ° C. </li></ol> 7. Grundlagen zur Schwimmblase <ol><li> Füllen Sie einen 10-ml-Spritze mit Hochvakuumfett. </li><li> Vorbereiten einer 2% LMA (Schritt 5.3 mit 160 mg des niedrig schmelzender Agarose in 8 ml E3). </li><li> Zu der gewünschten Zeit, betäuben die Fische (Schritt 5.2). </li><li> Übertragungs 10 Fische einer 1,7 ml-Zentrifugenröhrchen mit 1 ml E3 und euthanize durch Zugabe von 200 &amp; mgr; l 4 mg / ml Tricaine (Endkonzentration 800 ug / ml) in das Zentrifugenröhrchen und Inkubieren für 15 min bei Raumtemperatur. </li><li> Bereiten Sie eine Imaging-Objektträger, indem Sie 4 Tropfen Vakuumfett in einem Quadrat, 1 cm voneinander entfernt auf einem Mikroobjekt (75 x 25 mm, Abbildung 2). </li><li> Platzieren Sie 2 Tropfen 2% LMA in der Mitte des Platzes. </li><li> Setzen Sie einen Fisch auf einem separaten Glasobjektträger unter einem Binokular und überprüfen, ob das Herz hat aufgehört zu schlagen, entfernen Sie das überschüssige Wasser. </li><li&gt; Mit einer feinen Pinzette sezieren die Schwimmblase durch die Aufrechterhaltung der Kopf in einer festen Position mit dem linken Pinzette und Herunterziehen des vorderen Darm mit den richtigen Pinzette (4A). </li><li> Nehmen Sie den Schwimmblase durch den pneumatischen Kanal mit den linken Pinzette und trennt sie vom Verdauungstrakt, und legen Sie sie in der Mitte der 2% LMA, bevor es erstarrt. </li><li> Legen Sie ein Deckglas (18 x 18 mm) an der Oberseite des bildgebenden Glasplatte und schieben Sie es, bis es die Vakuumfett sowie die LMA (Abbildung 2) berührt. </li><li> Bild innerhalb von 10 Minuten der Dissektion, wie in Schritt 6.4 beschrieben. Wiederholen Sie die Schritte 7,7-7,11 mit den anderen Fischen. </li></ol>

The authors have nothing to disclose.

Fortschritte und Grenzen der Schwimmblase Mikroinjektion Krankheitsmodell Das hier vorgestellte Modell ist eine Erweiterung des in Gratacap et al Schleimhaut Candidiasis Tauch Modell (2013). es fügt die Vorteile eines kontrollierten Infektion Zeit eine hoch reproduzierbare Infektionsdosis und damit eine verbesserte Effizienz. Wir zeigen hier neue Methoden, die nicht-invasive zeitliche Dokumentation der Infektionsdynamik im Detail als auch eine höhere Auflösung Ex-vivo-B...

Mucosal infections can lead to life threatening bloodstream infections due to the damage of the epithelial barrier, which allows pathogens access to the systemic environment1,2. In addition, mucosal infections can also cause significant immunopathology even when contained externally3-5. The commensal unicellular fungus Candida albicans is present in the majority of the population in the oral cavity and other mucosal sites6-9. Although normally contained by innate and adaptive immune responses, innate immune defects and medical interventions can lead to severe mucosal candidiasis. The assault on the epithelial barrier results in an increased risk of life threatening disseminated disease as well as immunopathology, as in the case of vulvo-vaginal candidiasis, additionally C. albicans colonization has been linked with lung immune homeostasis10,11. Disseminated candidiasis is now the fourth most common bloodstream infection in intensive care units12 and mortality as high as 40% makes it a major concern. Due to the increase in immunomodulatory treatments for patients with autoimmune diseases, cancer or organ transplants, it is imperative to understand the interaction between this pathogen and the mucosal immune compartment.
The majority of cell biological advances regarding C. albicans-cell interactions at the mucosal level come from in vitro13-15 and murine models16-18. Both these approaches have distinct advantages, but the ability to image live cells at high resolution in an intact host has limited the temporal and spatial characterization of the infection. For these studies, there is the need for an in vivo model where the interaction of pathogen, innate immune and epithelial cells can be visualized in an intact vertebrate host.
The zebrafish has emerged as an invaluable tool for the understanding of human disease, mainly due to its transparency and amenability to genetic manipulation. Cell and organ development have been imaged in exquisite detail, which has led to the description of novel immune cell behaviors, such as T cell behavior in the developing thymus19 or the battle between intracellular mycobacteria and phagocytes20-22. Recent work has described intestinal microbe-host interactions in zebrafish and shown that microbial colonization of the intestinal tract affects host intestinal physiology and resistance to other infections23,24. Furthermore, infection through the gut epithelium has been described for several pathogens.
In contrast to the intestinal tract, the swimbladder represents a more isolated and complementary mucosal model. This organ is an extension of the developing gut tube and forms anteriorly to the liver and pancreas25,26. It produces surfactant, mucus and antimicrobial peptides27,28 and anatomically, as well as ontogenetically, this organ is considered a homologue of the mammalian lung29,30. Since the pneumatic duct remains connected to the gut in the zebrafish, this allows for immersion infection to occur naturally. Remarkably, the only known naturally occurring infections of fish with Candida species are C. albicans infections in the swimbladder31. We recently described an experimental immersion infection model where C. albicans infects the swimbladder, and found that this infection recapitulates some of the hallmarks of C. albicans-epithelial interaction in vitro32,33. 
In the method presented here, the original immersion infection model is improved by directly injecting C. albicans into the swimbladder of 4 days post fertilization (dpf) zebrafish. This allows for precise temporal control of infection as well as a highly reproducible inoculum. It permits detailed intravital imaging, coupled with the versatility of the zebrafish model. As an example of what can be done with this method, we present the spatio-temporal dynamics of C. albicans growth along with neutrophil recruitment to the site of infection. Because zebrafish swimbladder tissue is challenging to image intravitally, we also present a rapid swimbladder dissection technique that improves fluorescence signal and microscopic resolution. These methods expand the toolbox for fungal, immunological, and aquaculture research as well as describing a novel infection route that may be translated to model other fungal, bacterial or viral infections of mucosal surfaces.

<table border="0" cellpadding="0" cellspacing="0" width="784">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Name</td>
			<td style="width:259px;">Company</td>
			<td style="width:176px;">Catalog Number</td>
			<td style="width:88px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.7 mL tubes</td>
			<td>Axygen</td>
			<td>MCT-175-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deep Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>89107-632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>VWR Scientific</td>
			<td>90000-726</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone</td>
			<td>VWR Scientific</td>
			<td>90000-264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextrose</td>
			<td>Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>VWR Scientific</td>
			<td>90000-760</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine tweezers (Dumont Dumoxel #5)</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wooden Dowels</td>
			<td>VWR Scientific</td>
			<td>10805-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low Melt Agarose</td>
			<td>VWR Scientific</td>
			<td>12001-722</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flaming Brown Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate capillary</td>
			<td>Sutter Instruments</td>
			<td>BF120-69-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MPPI-3 Injection system</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MPPI-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Back Pressure Unit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>BPU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette Holder kit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MPIP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Foot Switch</td>
			<td>Applied Scientific Instrumentation</td>
			<td>FSW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MM33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnetic Base</td>
			<td>Applied Scientific Instrumentation</td>
			<td>Magnetic Base</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tricaine methane sulfonate</td>
			<td>Western Chemical Inc.</td>
			<td>MS-222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting Scope</td>
			<td>Olympus</td>
			<td colspan="2">SZ61 top SZX-ILLB2-100 base</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscope</td>
			<td>Olympus</td>
			<td colspan="2">IX-81 with FV-1000 laser scanning confocal system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20x microscope objective</td>
			<td>Olympus</td>
			<td>UPlanSApo 20x/0.75</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roller drum</td>
			<td>New Brunswick Scientific</td>
			<td>TC-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microloader pipette tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass culture tubes (16 x 150 mm)</td>
			<td>VWR Scientific</td>
			<td>60825-435</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>VWR Scientific</td>
			<td>BDH4534-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>VWR Scientific</td>
			<td>BDH4532-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4</td>
			<td>VWR Scientific</td>
			<td>BDH0246-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (Corning)</td>
			<td>VWR Scientific</td>
			<td>BDH4520-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Children clay (Play-Doh)</td>
			<td>Hasbro</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Fisher Scientific</td>
			<td>C69-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylene Blue</td>
			<td>VWR Scientific</td>
			<td>VW6276-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PTU</td>
			<td>Sigma</td>
			<td>P7629-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer (Hausser scientific)</td>
			<td>VWR Scientific</td>
			<td>15170-172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Type A immersion oil</td>
			<td>Blue Marble Products</td>
			<td>51935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex Genie</td>
			<td>VWR Scientific</td>
			<td>14216-184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose (Lonza)</td>
			<td>VWR Scientific</td>
			<td>12001-870</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fishing wire</td>
			<td>Stren</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well imaging plate (Sensoplate)</td>
			<td>Greiner Bio-One</td>
			<td>655892</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High vacuum grease (Dow Corning)</td>
			<td>VWR Scientific</td>
			<td>59344-055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microslide (25 x 75 mm)</td>
			<td>VWR Scientific</td>
			<td>48300-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover slips (18 x 18 mm), No 1.5</td>
			<td>VWR Scientific</td>
			<td>48366-045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 cm Petri dish (Olympus plastics)</td>
			<td>Genesee Scientific</td>
			<td>32-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol (EMD chemicals)</td>
			<td>VWR Scientific</td>
			<td>EMGX0185-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well culture dish (Olympus plastics)</td>
			<td>Genesee Scientific</td>
			<td>25-107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Weight boats (8.9 cm)</td>
			<td>VWR Scientific</td>
			<td>89106-766</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling mucosal candidiasis in larval zebrafish by swimbladder injection

Frühe Verteidigung gegen Krankheitserreger Schleimhaut besteht sowohl eine Epithelbarriere und angeborenen Immunzellen. Die immunocompetency von beiden, und ihre Zwischenverbindung, an erster Stelle stehen für den Schutz vor Infektionen. Die Wechselwirkungen von Epithelzellen und Zellen des angeborenen Immunsystems mit einem Pathogen besten in vivo, wo komplexe Verhalten entfaltet über Zeit und Raum untersucht. Allerdings sind bestehende Modelle nicht für den einfachen Raum-Zeit-Bildgebung der Kampf mit Krankheitserregern an der Schleimhautniveau ermöglichen. Die hier entwickelten Modell erstellt eine Schleimhaut-Infektion durch direkte Injektion des Pilzpathogen, Candida albicans, in die Schwimmblase der jugendlichen Zebrafisch. Die daraus resultierende Infektion ermöglicht hochauflösende Bildgebung von epithelialen und angeborenen Immunzellverhaltens in der gesamten Entwicklung der Mucosal Disease. Die Vielseitigkeit dieser Methode ermöglicht die Abfrage des Host, um die detaillierte Abfolge der Ereignisse, die zu Immun pH-Sondeagocyte Rekrutierung und die Rollen von bestimmten Zelltypen und molekulare Signalwege in Schutz zu untersuchen. Darüber hinaus kann das Verhalten des Erregers als Funktion der Immunangriff gleichzeitig unter Verwendung fluoreszierende Protein exprimierenden C. abzubildenden albicans. Erhöhte räumliche Auflösung der Wirt-Pathogen-Interaktion ist auch möglich, mit dem beschriebenen schnellen Schwimmblase Dissektionstechnik. Die hier beschriebene Schleimhaut-Infektionsmodell ist einfach und sehr gut reproduzierbar, so dass es ein wertvolles Werkzeug für die Untersuchung der Schleimhautinfektionen. Dieses System kann auch allgemein übersetzbar andere Schleimhautpathogene, wie beispielsweise mykobakteriellen, bakterielle oder virale Keime, die normalerweise durch epitheliale Oberflächen infizieren.

Mikroinjektion in der hinteren Schwimmblase Die hier präsentierten experimentellen Methode beschreibt die Injektion von einem konsistenten Dosis C. albicans Hefezellen in der Schwimmblase von 4 dpf Zebrafisch. Frühere Arbeiten mit der Immersions Modell schlägt vor, dass der Schwimmblase Immunantwort auf C. albicans ähnelt Säugerschleimhaut Candidiasis 32. Hier zeigen wir eine modifizierte Infektionsmethode, die einfacher, reproduzierbare und schnelle ist; mehre...

Watch this Scientific Journal Video about Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection at JoVE.com

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Modellierung Mucosal Candidiasis in Zebrafisch-Larven von Schwimmblase Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

modeling-mucosal-candidiasis-larval-zebrafish-swimbladder

Research

JoVE Journal

Immunology and Infection

10.1K Aufrufe.  University of Maine. In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Serie: Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Nicht-invasive Bildgebung von disseminierter Candidiasis in Zebrafischlarven

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Forschung

Immunologie und Infektion

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

In vitro Coculture Assay zur Bewertung pathogeninduzierter Neutrophiler Transepithelmigration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Nicht-invasive Bildgebung von der angeborenen Immunantwort in einem Zebrafisch-Larven Modell<em&gt; Streptococcus iniae</em&gt; Infektions

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

Nicht-invasive Probenahme von Schleimhaut-Flüssigkeit für die Quantifizierung von In Vivo oberen Atemwegen immun-Mediator-Level

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Galle-Salz-induzierte Biofilmbildung im enterischen Krankheitserregern: Techniken für die Identifizierung und Quantifizierung

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Modellierung von Tuberkulose Mycobacterium Marinum infizierten Erwachsenen Zebrafisch

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

Die Galleria Mellonella Waxworm Infektionsmodell für verbreitet Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

Induktion der Maus Lungenverletzung durch Endotracheal Injektion von Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells to inflammatory disease due to their functionally conserved innate immune system. They are also widely used to examine how innate immune cells help guide developmental processes. By taking advantage of the optical transparency and genetic tractability of larval zebrafish, these studies often focus on live imaging approaches to functionally characterize fluorescently marked macrophages and neutrophils within intact animals. Due to their diverse functional heterogeneity and ever-expanding roles in disease pathogenesis, macrophages have received significant attention. In addition to genetic manipulations, chemical interventions are now routinely used to manipulate and examine macrophage behavior in larval zebrafish. Delivery of these drugs is typically limited to passive targeting of free drug through direct immersion or microinjection. These approaches rely on the assumption that any changes to macrophage behavior are the result of a direct effect of the drug on the macrophages themselves, and not a downstream consequence of a direct effect on another cell type. Here, we present our protocols for targeting drugs specifically to larval zebrafish macrophages by microinjecting drug-loaded fluorescent liposomes. We reveal that poloxamer 188-modified drug-loaded blue fluorescent liposomes are readily taken up by macrophages, and not by neutrophils. We also provide evidence that drugs delivered in this way can impact macrophage activity in a manner consistent with the mechanism of action of the drug. This technique will be of value to researchers wanting to ensure targeting of drugs to macrophages and when drugs are too toxic to be delivered by traditional methods like immersion.

Here, we describe the synthesis of drug-loaded liposomes and their microinjection into larval zebrafish for the purpose of targeting drug delivery to macrophage-lineage cells.

Targeting Drogen zu Larval Zebrafish Makrophagen durch Injektion von Drogen-geladenen Liposomen

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Isolierung von Tonsillar Mononukleären Zellen zur Untersuchung von Ex-Vivo-angeborenen Immunantworten in einem menschlichen Mukosal-Lymphgewebe

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...