Diese Studie wurde von der United States Department of Agriculture, Agricultural Research Service unterstützt. Handelsnamen oder kommerzielle Produkte in dieser Veröffentlichung sind der Bereitstellung von spezifischen Informationen ausschließlich für den genannten Zweck und Empfehlung oder Billigung nicht durch die United States Department of Agriculture implizieren. USDA ist eine Chancengleichheit Anbieter und Arbeitgeber.

1. Der Peripheral Fringe von Bakterienkolonie <ol><li> Wachsen X. fastidiosa (Xf) Temecula Wildtyp 22, mutant Pilg Deletion Xf ΔpliG (unter Verwendung von zuvor beschriebenen Löschstrategie 23) und sein komplementäres XfΔpliG -C (unter Verwendung von zuvor Chromosoms basierte genetische Komplementierung Strategie 24 beschrieben) auf PD2 Medium Agarplatten 25 bei 28 ° C für 5-7 Tage. </li><li> Autoklav Cellophan (1 x 1 cm 2) in Wasser bei 121 ° C (249 ° F) für 15 min. Pick-up ein Stück Zellophan, lassen Sie das Wasser durch das Berühren einer Ecke Zellophan auf einem leeren Petrischale, legen sorgfältig das Zellophan über 15% der Agar-Oberfläche und der Luft trocknen. </li><li> Pick - up einzelne X. fastidiosa Kolonien mit sterilen Zahnstochern gerundet und Spot - Zellen aseptisch auf einem sterilisierten Blatt Cellophan auf die 15% der Agar - Oberfläche in den Agarplatten überlagert. Die Plattes bei 28 ° C für 2-3 Tage. </li><li> Überprüfen Sie die Rand Morphologie der Kolonien ein Binokular mit einem 2X Objektiv und einem 10fach Augenlinse verwendet wird. Fotografieren Sie das periphere Rand um Kolonien. </li></ol> 2. Mikroskopie und Mikrofluidik Strömungskammern <ol><li> Fabrizieren Mikrofluidik - Vorrichtungen unter Verwendung photolithographischer Verfahren ähnlich den zuvor beschriebenen 5,18. Entwerfen vier parallele Kanäle mit computergestützten Design - Software auf dem Master - Silizium - Wafer unter Verwendung von Standard - Lithographieverfahren 26. </li><li> Erstellen Sie die mikrofluidischen Kammern aus Silizium-Wafer-Master mit Polydimethylsiloxan (PDMS). Pour unpolymerisierten PDMS über dem Siliziumwafer Master und härten bei 60 ° C für 1 Stunde. Ziehen Sie die PDMS-Replik aus dem Wafer-Master und schneiden Sie die PDMS-Nachbildung mit einer Klinge in einem 22 mm x 40 mm als die gleiche Größe eines Deckglas ab. </li><li> Setzen Sie die PDMS - Replik, ein Deckglas (22 x 40 mm 2), und eine Microscope Schieber (51 x 76 mm 2) an der Luft - Plasma bei 30 W für 2 min 27. Sandwich der PDMS Körper zwischen dem Deckglas und dem Glasmikroskop des Mikro fluidischen Kammer zu bauen. </li><li> Bohren Sie ein Loch (5,5 mm Durchmesser) durch die PDMS an jedem Ende des gemusterten-Kanal. Schneiden Sie den Silikongummischlauch in 12-20 cm lang. Stecken Sie ein Ende des Silikongummischlauch (5,1 mm Außendurchmesser, 2,1 mm Innendurchmesser, 0,8 mm Wand) in jede Öffnung Ende der Kanäle des PDMS-Replik, und verschließen Sie diese mit unpolymerisierten PDMS bei 60 ° C für 1 Stunde. </li><li> Schließen Sie das andere Ende des Schlauchs am stacheligen Ende der Kunststoff-Luer-Anschlüsse. Wickeln der zusammengebauten mikrofluidischen Kammern mit der Aluminiumfolie und autoklaviert werden sie für 20 min. </li><li> Sammeln Bakterienzellen von X. fastidiosa Wildtyp, Mutante Xf ΔpliG und komplementäre XfΔpliG -C über Schaben, Einweg - Impf mit Loops aus PD2 Mediumplatten. Passen Sie die Zelldichtezu einer optischen Dichte von 0,05 in PD2 Brühe bei 600 nm wie zuvor beschrieben 23. Sammeln Sie die Bakterienzelllösung in eine 1 ml Spritze Gasdichte. </li><li> Montieren Sie die Mikrofluidik-Vorrichtung auf einem inversen Mikroskop Bühne. Schließen Sie ein Einlassrohr zum 5 ml Gasdichte Spritze mit PD2 Brühe. Den 5 ml Spritze Gasdichte mit den Spritzenpumpen. </li><li> Verbinden Sie den Abflussrohr in einen Abfallbehälter. Pflegen eine mittlere Strömungsgeschwindigkeit von 0,2 &amp; mgr; l min -1 für 30 min zur Stabilisierung des Systems. </li><li> Verbinden Seiteneinlaßrohre an eine 1 ml Spritze, die die Gasdichte Bakterienzelllösung enthält. Spülen Sie die bakterielle Zelllösung durch den Gummischlauch, bis der Kanal erreicht ist. Pflegen Sie eine mittlere Strömungsgeschwindigkeit von 0,2 &amp; mgr; l min -1 für weitere 30 Minuten , um ungebundene Zellen aus der Kammer vor der Bildaufnahme zu spülen. </li><li> Bringen Sie den Mikroskop Verschluss unter dem Feld angepasst Teil des Mikroskops das Licht zu steuern. Schließen Sie den Verschluss auf den Auslöser control System und eine Verbindung mit dem Computer das Rolltor-Steuerungssystems. </li><li> Montieren Sie eine Digitalkamera an den Videoanschluss des Mikroskops und verbinden Sie es mit dem Computer. Führen Sie die Zeitraffer-Aufnahme-Software, wählen Sie die &quot;Shutter&quot; Funktion aus dem Menü, und erkennen automatisch die installierte Shutter als Standard in der Software-Verbindungen mit dem Verschluss mit der Software zu etablieren. </li><li> Wählen Sie die &quot;Digitalkamera&quot; Funktion aus dem Menü des Zeitraffer-Aufnahme-Software, um automatisch die Digitalkamera als Standard-Capture-Gerät in der Software erkennen und die Kommunikation mit der Digitalkamera mit der Software. </li><li> Suchen Sie die Bakterienzellen in einem der Kanäle 20x Phasenkontrast-Optik verwenden, schalten dann auf die 40-fach Objektivlinse vor der Bilderfassung. </li><li> Führen Sie die Zeitraffer-Aufnahme-Software, wählen Sie die &quot;Bildaufnahme&quot; -Funktion Standardparameter aus dem Menü die Bilder aus dem Mikroskop zu erwerben.Dann öffnen Sie den &quot;Acquire Zeitraffer-Funktion&quot; und auf 30 sec 5,18,28 für die Dauer von 6-24 Stunden , je nach Experiment benötigt Zucken Motilität von X. das Zeitintervall zu beobachten fastidiosa 5,18,28. Klicken Sie auf &quot;OK&quot;, um die Zeitraffer-Aufnahme zu starten. Klicken Sie auf &quot;Stapelfunktion&quot; aus dem Menü wählen Sie &quot;Speichern unter&quot; die Bilder im Zielordner auf dem Computer zu stapeln, nachdem die Aufnahme wird beendet. </li><li> Für mehrere Kanäle, Zeitraffer-Bilder aus dem ersten Kanal erfassen alle 30 Sekunden für 6 Stunden. Bewegen der Objektivlinse des Mikroskops auf den nächsten Kanal der Zielzellen zu lokalisieren. Wiederholen Sie die Zeitraffer-Funktion wie oben beschrieben, Bilder in jedem der vier Kanäle sequentiell zu erfassen, wenn Experiment wird bis zu vier Kanäle nutzen. Fortsetzung der Zeitraffer-Bildaufnahme für so lange wie drei aufeinander folgenden Tagen. Alle Experimente wurden bei Raumtemperatur (23 ± 2 ° C) durchgeführt. </li><li> Stellen Sie die Zeitraffer-Bilder in einVideodatei die Zeitrafferaufnahme Imaging-Software. Führen Sie Zeitraffer-Aufnahme-Software, klicken Sie auf &quot;Stapelfunktion&quot; aus dem Menü, und wählen Sie &quot;Open-Stack-Funktion&quot;, um die gestapelten Dateien vom Computer öffnen. </li><li> Starten Sie den &quot;Make Movie&quot; Funktion aus dem Stack-Modul, alle Bilder auswählen und das &quot;AVI&quot; Ausgabeformat auswählen. Klicken Sie auf &quot;Speichern unter&quot; auf dem Computer die Videodatei im Zielordner zu speichern. </li><li> Wählen Sie die kompilierten Filme aus dem Zielordner auf dem Computer und spielen sie. Dann beobachten die Beweglichkeit der einzelnen Zellen durch die resultierende Visualisierung der Zucken Motilität Aktivität von Bakterienzellen in den erzeugten Videodateien. </li></ol>

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Die Autoren erklären, dass sie keine finanziellen Interessen haben.

In dieser Studie charakterisierten wir das Bewegungsverhalten von X. fastidiosa Pilg Mutante Xf ΔpilG und seinen komplementären XfΔpilG- C - Stämme in neu mehrere parallel-Nano-Kanal Mikro fluidischen Kammern ausgelegt. Die neu entwickelten Mikro fluidischen Kammern können bis zu vier parallelen Kammern haben mit 100 &amp; mgr; m Nanokanal in der Breite im Vergleich zu früheren Konstruktionen mit nur einem einzigen 50 &amp; mgr; m breiten Kanal 18. Die verbes...

Xylella fastidiosa ist ein Gram-negative nicht gegeißelt, pathogene Bakterium , das eine Reihe von wirtschaftlich wichtigen Pflanzenkrankheiten verursacht, einschließlich Pierce-Krankheit in Weinrebe (Vitis vinifera L.) 1,2, 3. Dieses Bakterium ist begrenzt auf den wasserführenden Xylem Schiffe. Die Infektion der Weinrebe bewirkt , dass die Blockade der Xylemgefäße und führt zu Wasserstress und Mangelernährung 3. Erfolgreiche Besiedelung hängt von der Fähigkeit des Bakteriums von der anfänglichen Stelle der Infektion mit dem Rest der Anlage 3 zu bewegen. Zucken Motilität ist ein Mittel der Flagellen-unabhängige Bakterien Bewegung durch die Erweiterung, Anhaftung und das Zurückziehen des polaren Typ IV - Pili 4 , die in X charakterisiert wurde fastidiosa 5,6,7. Die Zuckungen Motilität wurde durch Laser - Pinzette und Rasterkraftmikroskopie (AFM) 8,9,10 beobachtet. Unter Verwendung dieser Techniken twitching Motilitäten von Typ - IV - Pili von N. erzeugt gonorrhoeae und P. aeruginosa wurden durch fl uorescently Kennzeichnung Pili charakterisiert und ihre Bewegungen mikroskopisch zu erfassen. Obwohl beide Verfahren die Haftkraft der einzelnen Bakterien detailliert sind, sind die Verfahren kompliziert und zeitraubend 9,10. Die Mikro fluidischen Kammern wurden verwendet , 5,6 Fern Migration von einzelnen Zellen sowie kleine Aggregate von Bakterienzellen zu beobachten. Diese Kammern wurden als mikrostrukturierte -Nano-Kanal in einer Platte gestaltet , integriert mit einem Zeitraffer-Bildaufnahmesystem 11,12,13,14. Micro fluidischen Kammer Geräte mehrere Vorteile für die Untersuchung der Bewegungsverhalten und Zell-Zell-Wechselwirkungen von Bakterien bieten: (i) es stellt eine integrierte Plattform mit Mehrkanal-Fähigkeiten; (Ii) kann es die Bewegungen und Aggregationen von einzelnen Zellen in den nanoskaligen Eigenschaften von Bakterien zu untersuchen; (Iii) es erlaubt die direkte microscopic Bildaufzeichnung von Bakterienzellen und Zeitraffer-Analyse, (iv) es bietet langfristige räumliche und zeitliche Beobachtungen einzelner und / oder Populationen von Bakterien in einer Mikro-Umgebung; (V) die Strömungsgeschwindigkeit des Kulturmediums in einen Kanal genau gesteuert werden kann, und (vi) nur ein sehr kleines Volumen (1 ml) des Kulturmediums wird für jedes Experiment erforderlich. Vor kurzem hat die Mikro fluidischen Strömungssystem wurde das Verhalten von Bakterienzellen unter verschiedenen Mikroumgebungen 14,15,16 zu untersuchen , beschäftigt. Die Haftfestigkeit und die Oberflächenbefestigung von E. coli 15, X. fastidiosa 16 und Acidovorax citrulli 14 auf Glasoberflächen wurden unter Verwendung von Mikro fluidischen Kammern beurteilt. Die Aggregation und Biofilm - Bildung durch Typ IV - Pili von Acidovorax citrulli vermittelt wurden 14 analysiert. Ferner kann die Bewegung der A. citrulli beobachtet unter fl ow cie Bedingungen gezeigt , dass der Typ IV - Pili eine wichtige Rolle bei der Besiedlung spielen kann und von A. verbreiten citrulli in Xylemgefäße unter dem Splint fl ow Bedingungen. Die Zuckungen Motilitäten von Pseudomonas aeruginosa und X. fastidiosa Zellen wurden gegen einen Flüssigkeitsstrom in einer mikrofabrizierten Strömungskammer 5,6,17 erfolgreich beobachtet. Typ - IV - Pili - defizienten pilB und pilQ Mutanten von X. fastidiosa wurden 5,6,18 die Geschwindigkeit der Zucken Motilität unter den Strömungsbedingungen in Mikro fluidischen Geräten zu tiefgreifend verändern gefunden. Die Studien , die auf bakterielle Adhäsion und Motilität in Mikro fluidischen Vorrichtungen zeigten , dass die Mikro fluidischen Kammern sind besonders geeignet für die Analyse der Zuckungen Motilität und Migration von Pili-vermittelten Bakterien in vitro. Diese Ergebnisse erklären die Zuckungen-vermittelte Migration Mechanismus, der Zell-Zell-Bindung erleichtert, Aggregation und Kolonisierung innerhalbder Wirt, führen schließlich zu einer systemischen Infektion. Pil-CHP - Operon von X. fastidiosa enthält Pilg, Pili, pilJ, pille, chpB und KWKK die Transduktion kodieren Signal 20 Pfade. Die Transchemorezeptoren binden chemische Reize in den periplasmatischen Domäne und aktivieren eine Signalkaskade in ihrem Cytoplasma-Teil, um schließlich bakterielle Zucken Motilität kontrollieren. Im Pil-CHP - Operon von X. fastidiosa, ein Phospho-Shuttle - Protein Pilg ist ein Homolog zu CheY. In E. coli und P. aeruginosa, ist CheY die Antwortregulator in Chemotaxis - Systeme , die 19, 21 mit den Geißeln Motorproteine ​​interagieren. Obwohl die Beiträge des Pil-Chp Operon zu Virulenz in X. fastidiosa wurden kürzlich 20, die Rolle der Pilg in Chemotaxis - Operon in Reaktion auf die Umgebungssignale und geregelt / Motor Typ IV - Pili von X. sucht fastidiosa ist unclear. Um die Einsicht der Chemotaxis Regler Pilg in der Aktivität der Zucken Motilität von X. aufzuklären fastidiosa, einem Mikro fluidischen Kammer verwendet , um die Zuckungen Motilität von X. zu beurteilen fastidiosa. Die Pilg von X. fastidiosa wird durch den Vergleich der Phänotypen einer Deletionsmutante Xf ΔpliG, komplementären Stamm XfΔpliG -C und ihrem Wildtyp in vitro charakterisiert. Die Ergebnisse unterstreichen die Rolle der Pilg im Zucken Motilität von X. fastidiosa.

<table>
	<tbody>
		<tr>
			<td>Biology materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X. fastidiosa (Xf) Temecula wild type</td>
			<td></td>
			<td></td>
			<td>Costa, H. S., et al., 2004 22</td>
		</tr>
		<tr>
			<td>pilG deletion mutant Xf&#916;pliG</td>
			<td></td>
			<td></td>
			<td>Shi, X. Y., et al., 2007 26</td>
		</tr>
		<tr>
			<td>pilG complementary strain Xf&#916;pliG-C&#160;</td>
			<td></td>
			<td></td>
			<td>Davis, M. J., wt al. 1998 23</td>
		</tr>
		<tr>
			<td>Physical materials and equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable inoculating loops</td>
			<td>VWR international, Radnor, PA</td>
			<td>#22-363-607</td>
			<td>quantitative procedures such as bacterial collection</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning Corporation</td>
			<td>#0002709226</td>
			<td>Sylgard 184 silicone Elastomeric Kits</td>
		</tr>
		<tr>
			<td>AmScope MD2000 digital camera</td>
			<td>AmScope, Irvine, CA</td>
			<td>SE305R-AZ-E</td>
			<td>Image, video recording and measurement&#160;</td>
		</tr>
		<tr>
			<td>Tubes line</td>
			<td>Edgewood, NY</td>
			<td>#T4300</td>
			<td>Connected to the syringe and microfluidic chamber</td>
		</tr>
		<tr>
			<td>Plastic luer connectors</td>
			<td>Edgewood, NY</td>
			<td></td>
			<td>Connected to the syringe and microfluidic chamber</td>
		</tr>
		<tr>
			<td>Syringe pumps</td>
			<td>Pico Plus, Harvard Apparatus, MA</td>
			<td>#702209</td>
			<td>The flow rate can be adjusted while the pump is running.</td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>Gastight, Hemilton Company, Reno, NV</td>
			<td>#1005</td>
			<td>Provide the flowing broth</td>
		</tr>
		<tr>
			<td>Inverted Olympus IMT-2 microscope</td>
			<td>Olympus</td>
			<td>IMT-2 FLuoro PHase</td>
			<td>Image observation and recording</td>
		</tr>
		<tr>
			<td>SPOT-RT digital camera</td>
			<td>Diagnostic Instruments, Inc., MI</td>
			<td>RT230</td>
			<td>Image, video recording and measurement</td>
		</tr>
		<tr>
			<td>Microscope Shutter</td>
			<td>The UNIBLITZ, US</td>
			<td>#LS2T2</td>
			<td>Control camera&#8217;s exposure time</td>
		</tr>
		<tr>
			<td>Microscope Shutter Control system</td>
			<td>The UNIBLITZ, US</td>
			<td>VCM-D1</td>
			<td>VCM-D1 Single Channel CE/UL/CSA Approved Shutter Driver</td>
		</tr>
		<tr>
			<td>MetaMorph Image software</td>
			<td>Universal Imaging Corp., PA</td>
			<td></td>
			<td>Real-time super-resolution image processing&#160;</td>
		</tr>
	</tbody>
</table>

visualization of twitching motility and characterization of the role of the pilg in xylella fastidiosa

Xylella fastidiosa ist ein Gram-negative nicht-flagellated Bakterium , das eine Reihe von wirtschaftlich bedeutenden Pflanzenkrankheiten verursacht. Die Zuckungen Motilität bietet X. fastidiosa ein Mittel für den Ferninnerbetrieblichen Bewegung und Kolonisation, einen Beitrag zu Pathogenität in X. fastidiosa. Die Zuckungen Motilität von X. fastidiosa wird von Typ IV - Pili betrieben. Typ IV - Pili von Xylella fastidiosa werden durch Pilg geregelt, einem Chemotaxis - Regler in Pil-Chp Operon für Proteine ​​kodiert , die mit Signaltransduktionswege beteiligt sind. Um die Rolle von Pilg im Zucken Motilität von X. aufzuklären fastidiosa, ein Pilg defizienten Mutante Xf ΔpilG und seinen komplementären Stamm XfΔpilG- C enthält nativen Pilg entwickelt. Ein mikrofluidischen Kammern mit einem Zeitraffer-Bildaufnahmesystem integriert wurde verwendet , Zucken Motilität in XfΔp zu beobachtenILG, XfΔpilG- C und seine Wildtyp - Stamm. Unter Verwendung dieses Aufzeichnungssystem ermöglicht es langfristige räumliche und zeitliche Beobachtungen der Aggregation, Migration einzelner Zellen und Populationen von Bakterien über Zucken Motilität. X. fastidiosa Wildtyp und komplementäre XfΔpilG- C - Stamm zeigte typische Zucken Motilität Eigenschaften direkt in den mikrofluidischen Strömungskammern beobachtet, während Mutante XfΔpliG die Zuckungen defizienten Phänotyp aufwiesen. Diese Studie zeigt , dass Pilg zur twitching Motilität von X. trägt fastidiosa. Die mikrofluidischen Strömungskammer für das Beobachten twitching Motilität als Mittel verwendet.

Das Vorhandensein einer peripheren Kolonie fringe indikativ für Typ IV - Pilus-vermittelten twitching Motilität wurde in den Kolonien von X. beobachtet fastidiosa Wildtyp und komplementäre Xf ΔpliG -C Stamm (Abbildung 1). Mutant XfΔpliG zeigen jedoch nicht Fransen um den Umfang der Kolonien (Abbildung 1). Zeitraffer-Bildgebung von Bakterienzellen in Nano mikrofluidischen Strömungskammern ergab , dass Zuc...

Watch this Scientific Journal Video about Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa at JoVE.com

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

In dieser Studie wurde ein Nano mikrofluidischen Strömungskammer wurde verwendet , um die Zuckungen Motilität von Xylella fastidiosa zu visualisieren und funktionell zu charakterisieren, ein Bakterium , das Pierce-Krankheit in Weinrebe verursacht.

Visualisierung von Twitching Motilität und Charakterisierung der Rolle des<em&gt; Pilg</em&gt; in<em&gt; Xylella fastidiosa</em

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility ...

The over goal of this protocol is to functionally characterize the genes responsbile for the twitching motility of bacteria using a nano microfluidic flow chamber integrated with a time-lapse image recording system.This method can help answer key questions about a bacteria phenotype and associated gene function, such as the twitching motility of bacteria.The main advantage of this technique is that it provides a special and a tempered observation for aggregation migration of individual cells and populations.Demonstrating this procedure will be Dr.Xiangyang Shi, a post-doc from my laboratory.Using a standardized deletion strategy, grow Temecula wild-type Xylella fastidiosa, grow pilG deletion mutants, and grow the complimentary strain all on PD2 medium agar plates.Incubate the bacteria at 28

visualization-twitching-motility-characterization-role-pilg-xylella

Research

JoVE Journal

Immunology and Infection

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

10.6K Aufrufe.  University of California, Davis. The over goal of this protocol is to functionally characterize the genes responsbile for the twitching motility of bacteria using a nano microfluidic flow chamber integrated with a time-lapse image recording system.This method can help answer key questions about a bacteria phenotype and associated gene function, such as the twitching motility of bacteria.The main advantage of this technique is that it provides a special and a tempered observation for aggregation migration of individual cells ...

Serie: Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-&alpha; knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

Überprüfung der Rolle der Nasopharynx-assoziierte lymphoide Gewebe (NALT) in Maus auf Impfstoffe

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

Forschung

Immunologie und Infektion

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Eine quantitative Auswertung der Zellmigration durch die Phagokinetic Spur Motilität Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Qualitative und quantitative Assays zur Detektion und Charakterisierung von Protein Antimikrobielle

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Eine schnelle Methode zur Blutverarbeitung die Ausbeute an Plasma-Peptidkonzentrationen im menschlichen Blut zu erhöhen

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

Assays für die Untersuchung der Rolle von Vitronectin in bakterielle Adhäsion und Serum-Widerstand

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...

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 Extracellular Vesicles Produced by Iron-limited Mycobacteria

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) containing immunologically active molecules. Knowledge regarding the molecular mechanisms of vesicle biogenesis, the content of the vesicles, and their functions at the pathogen-host interface is very limited. Addressing these questions requires rigorous procedures for isolation, purification, and validation of EVs. Previously, vesicle production was found to be enhanced when M. tuberculosis was exposed to iron restriction, a condition encountered by Mtb in the host environment. Presented here is a complete and detailed protocol to isolate and purify EVs from iron-deficient mycobacteria. Quantitative and qualitative methods are applied to validate purified EVs.

Mycobacterium tuberculosis shows increased production and release of extracellular vesicles in response to low iron conditions. This work details a protocol for generating low iron conditions and methods for the purification and characterization of mycobacterial extracellular vesicles released in response to iron deficiency.

Isolierung und Charakterisierung von extrazellulären Vesikeln, die von eisenbegrenzten Mykobakterien hergestellt werden

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) ...

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

Visualisierung, Quantifizierung und Kartierung von Immunzellpopulationen in der Tumormikroumgebung

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Visualisierung der bakteriellen Resistenz mit fluoreszierenden Antibiotikasonden

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be altered by severe brain injury. This is evidenced by the fact that pneumonia is a major cause of mortality in patients who have suffered from ischemic stroke. The goal of this protocol is to describe the use of multicolor flow cytometric analysis to identify 13 types of immune cells in the lungs of mice, including alveolar macrophages, interstitial macrophages, CD103+ or CD11b+ dendritic cells (DCs), plasmacytoid DCs, eosinophils, monocytes/monocyte-derived cells, neutrophils, lymphoid-derived&#160; T and B cells, NK cells, and NKT cells, following ischemic stroke induction by transient middle cerebral artery occlusion. Moreover, we describe the preparation of lung homogenates using a bead homogenization method, to determine the expression levels of 13 different cytokines or chemokines simultaneously by multiplex bead arrays coupled with flow cytometric analysis. This protocol can also be used to investigate the pulmonary immune response in other disease settings, such as infectious lung disease or allergic disease.

This protocol describes the use of flow cytometry to identify the changes in immune cell composition, cytokine profile, and chemokine profile in the pulmonary environment following transient middle cerebral artery occlusion, a murine model of ischemic stroke.

Charakterisierung von Immunzellen und proinflammatorischen Mediatoren im pulmonalen Milieu

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be ...