The authors thank Ian Clarke and P. Scott Hefty for the pGFP-SW2 and pASK-GFP-mKate2-L2 plasmids, respectively. We thank Paul Miller for technical assistance and Richard Stephens for other resources. This work was funded by NIH grant AI095603 (KH).

1. Erzeugung von Leuchtstoff-Wirtszelllinien <ol><li> Tag 1. Platte 293T-Zellen (oder andere retrovirale Verpackungslinie) auf 6-Well-Platten mit 2 × 10 6 Zellen / Vertiefung, zu sein ~ 75% konfluent am nächsten Tag. Falls gewünscht, um die Platte zwei Vertiefungen für jede Retrovirus verwendet werden. </li><li> Tag 2. Saugen Sie Zellen und 2 ml frisches Wachstumsmedium (DMEM + 10% FBS + 2 mM L-Glutamin). Transfektion von Zellen mit 5-8 &amp; mgr; g jedes der retroviralen Vektor (enthaltend GFP) und Verpackungsvektor (z. B. pVSVg) unter Verwendung von Lipofectamine 2000 oder einem ähnlichen Reagenz, und nach den Anweisungen des Herstellers. </li><li> Inkubieren Zellen mit DNA für 4-6 Stunden in einem 37 ° C CO 2 -Inkubator und anschließend Ersetzen Medium mit 1 ml Wachstumsmedium. </li><li> Inkubieren Zellen für 48 Stunden in 37 ° C CO 2 -Inkubator. </li><li> Tag 3. Platte Zielzellen (z. B. HeLa) auf Platten mit 6 Vertiefungen bei einer Dichte von etwa 10 5 Zellen / Vertiefung sein ~ 50% konfluentnächster Tag. Stellen Sie sicher, positive Transfektion in 293T-Zellen durch die Überwachung GFP-Fluoreszenz auf einem inversen Mikroskop. </li><li> Tag 4. Sammeln Retrovirus enthaltende Überstände abgehoben 293T-Zellen mit einer Pipette und Den Überstand durch ein 0,45 um-Celluloseacetat-Spritzenfilter. </li><li> Entfernen Sie Medium aus HeLa-Zellen und 0,5 ml Wachstumsmedium, das 16 ug / ml Polybren. Hinzufügen ~ 0,5 ml filtriert Retrovirus an Zellen tropfenweise und inkubieren Zellen in einem 37 ° C CO 2 Inkubator für 24 Stunden. Speichern alle verbleibenden Retrovirus (dh aus dem Bohrloch zu duplizieren) bei 4 ° C. </li><li> Tag 5. Wenn zwei Vertiefungen wurden vorgenommen, um zusätzliche Retrovirus, wiederholen Sie Schritt 1.7 zu erzeugen, die in Reihe mit den übrigen retrovirale Partikel zu infizieren. </li><li> Tag 6. Passage transfizierten HeLa-Zellen 1: 1 in einem 10 cm Schale, die Auswahl-Antibiotika (zB 500 ug / ml Neomycin). </li><li> Warten Sie ausreichend Zeit für die transduzierten Zellen in Anwesenheit von Selektions antibio wieder wachsenTiC, wonach Zellen können durch Standard-Techniken mit einer geringeren Konzentration an Selektionsantibiotikum vermehrt werden (z. B. 200 ug / ml Neomycin). Anmerkung: Die Zellen können auch klonal für optimale Helligkeit zu dieser Zeit ausgewählt werden, wenn nötig. </li></ol> 2. Erzeugung von GFP-exprimierenden Chlamydia trachomatis <ol><li> Vorbereitung 2x CaCl 2-Puffer (20 mM Tris pH 7,4, 100 mM CaCl 2) und 4SP Puffer (0,4 M Saccharose, 16 mM Na 2 HPO 4). </li><li> Tag 1. Tauen Sie gefrorenes C. trachomatis Lager auf Eis und sofort zu verdünnen 10 ul Bakterien in 50 ul 2x CaCl2 Puffer. In 3 ug Plasmid-DNA (z. B. pASK-GFP-mKate2-L2), gut mischen und Inkubation für 30 min bei 25 ° C. </li><li> Während dieser Stufe herzustellen Wirtszellen durch Trypsinierung eine T-75-Kolben McCoys-Zellen in einem Zentrifugenröhrchen. Zentrifugieren Sie die Zellsuspension bei 50 xg für 5 min und den Überstand verwerfen. Resuspend Zellpellet in geeigneten Volumen 1x CaCl2 Puffer zu einer Endkonzentration von 4 x 10 7 Zellen / ml zu ergeben. </li><li> Nach der 30 min Inkubation (aus Stufe 2.2) wird mit 100 &amp; mgr; l an Transformationsgemisch Rohr (Gesamtvolumen 200 ul) hergestellt McCoy-Zellen und Inkubation weitere 20 min bei 25 ° C. Zugabe von 100 ul dieser transformierten Zellgemisches auf eine einzelne Vertiefung einer Platte mit 6 Vertiefungen und Überlagerung mit 2 ml Wachstumsmedium. Inkubieren bei 37 ° C in einem CO 2 -Inkubator für 2 Tage. </li><li> Tag 3. Lyse McCoy Zellen mit C infiziert trachomatis durch Ersatz-Medium mit 1 ml dH 2 O und Kratzen Zellen mit einem gebogenen 1.000 ul Pipettenspitze. 1 ml 4SP Puffer und übergeben Suspension durch eine 27,5 G-Nadel ~ 5 mal für eine effiziente Zelllyse und Freisetzung von C. trachomatis. </li><li> Übertragungs 2 ml des Zellysats, die C. trachomatis in eine T-75-Kolben mit einer konfluenten Monolayer von frisch plattierten McCoy Zellen; 8 ml DMEM (ohne FBS) und Inkubation für 2 Stunden bei 25 ° C bis C ermöglichen trachomatis, sich an Zellen zu haften. </li><li> Saugen Sie Zellen und fügen Sie frisches Wachstumsmedium mit 10 U / ml Penicillin G und 1 ug / ml Cycloheximid ergänzt. Kolben für 48 h Inkubieren bei 37 ° C CO 2 -Inkubator. </li><li> Tag 4. Stellen Sie sicher, durch Lichtmikroskopie, die Zellen infiziert und Einschlüsse enthalten anomale Chlamydia. Identifizieren Einschlüsse so hell Vakuolen auf einer Standard-Lichtmikroskop, und anomale Chlamydia Körper als ringartige Objekte innerhalb Einschlüsse, die größer als 2 &amp; mgr; m Durchmesser. </li><li> Tag 5. Lyse Kultur durch Ersatz-Medium mit 2 ml dH 2 O und kratzen Zellen in einer Suspension. 2 ml 4SP Puffer und übergeben Suspension durch eine 27,5 G-Nadel ~ 5 mal. </li><li> Verwenden Sie 2 ml Zelllysat, einen frischen Monoschicht von McCoy-Zellen in einem gut in einer 6-Well-Platte zu infizieren. Zellen mit Lysat Inkubieren für 2 Stunden bei 25 ° C, absaugen und fügen Sie frisches WachstumsMedium, das Penicillin G und Cycloheximid. </li><li> Inkubieren Zellen in einem 37 ° C CO 2 -Inkubator für 3-5 Tage in Abhängigkeit von dem allgemeinen Gesundheitszustand der Zellen. </li><li> Wiederholen Sie die Schritte 2,9-2,10 einmal oder mehrmals, falls erforderlich, Passagieren Kulturen an die frische Wells in einer 6-Well-Platte, bis die normalen Suche Einschlüsse (ohne anomale Stellen) entstanden. Zu dieser Zeit verwenden einen invertierten Fluoreszenzmikroskop, um zu überprüfen, dass C. trachomatis auszudrücken mKate2 (rot). </li><li> Scale-up infizierten Kulturen zur Erzeugung von hohem Titer Chlamydienvorräte, beispielsweise durch Ernten Chlamydia Anten aus infizierten McCoy oder HeLa-Zellen in T-150 Kolben gezüchtet. </li></ol> 3. Infektion von Zellen mit Chlamydia <ol><li> Platte GFP-HeLa-Zellen auf Wunsch Kulturgefäß, zum Beispiel Glasbodenschalen oder Kammer-Objektträgern für die hochauflösende Bildgebung, oder 24-Well-Platten für IFU Entschlossenheit und Multiwell-Screening. </li><li> Auftauen frozen Röhrchen mit C. trachomatis, C. muridarum, oder C. pneumoniae auf Eis und verdünnter in HBSS auf gewünschte Multiplizität der Infektion (MOI), beispielsweise MOI = 1. </li><li> Führen entweder statisch oder Zentrifugation gestützten Infektionen bezogen auf die Chlamydia-Stamm, der verwendet wird. <ol><li> Bei Infektionen mit C. trachomatis LGV Serovar L2 oder C. muridarum waschen GFP-HeLa-Zellen mit HBSS und Inkubation mit verdünntem Bakterien für 2 h bei 25 ° C. Absaugen und waschen Sie die Zellen mit HBSS, und inkubieren Zellen in Wachstumsmedium in 37 ° C CO 2 Inkubator. </li><li> Bei Infektionen mit C. trachomatis Serovar D oder C pneumoniae, waschen GFP-HeLa-Zellen mit HBSS und fügen verdünnt Bakterien-Zellen. Zeigen Multiwellplatte oder Glasbodenschale (vom Band in einer Multiwellplatte Deckel gesichert) in einer Multiwellplatte Inhaber einer Schwingbecherrotor Anlage in einer Tischzentrifuge. </li><li> Zentrifuge Zellen bei 900 g bei 25 ° C für 1 Stunde. </li&gt; <li> Entfernen Kulturgefäße aus der Zentrifuge und in eine 37 ° C CO 2 Inkubator für 1 Stunde. </li><li> Saugen Sie Zellen in einer Biosicherheitswerkbank, waschen Sie die Zellen zweimal mit HBSS, und fügen Sie frisches Wachstumsmedium. Ortszellen in einem 37 ° C CO 2 -Inkubator. </li></ol></li></ol> 4. Live Cell Visualisierung von Chlamydia Einschlüsse <ol><li> Entfernen Chlamydia infiziert GFP-HeLa-Zellen, die aus CO 2 Inkubator und mit RPMI ersetzen Medium ohne Phenolrot + 5% FBS. </li><li> Berg Zellen auf einem invertierten Fluoreszenzmikroskop (Nikon Eclipse-Ti-E oder ähnliches) mit einem 20-fach oder höher Öl-Objektiv. </li><li> Verwenden von Imaging-Software (Volocity 6 oder ähnliches), konzentrieren sich auf und identifizieren Chlamydia infiziert GFP-HeLa-Zellen: grüne Zellen, die große schwarze Löcher (Chlamydia Einschlüsse). </li><li> Markieren Sie die xyz Positionen der Regionen, die Chlamydia infizierten Zellen von Interesse unter Verwendung von Imaging-Software (Volocity 6 oder ähnliches). Führen Sie dies durch manuelles &quot;Hinzufügen von Punkten&quot;, während in der XY-Bühne Ansichtsmodus. Auf diese Weise werden XYZ-Koordinaten für jeden markierten Stelle gespeichert. </li><li> Im Dialogfeld Acquisition, zu konfigurieren Software auf grün (GFP, HeLa Cytosol) und rot (mKate2, C. trachomatis) Kanälen und mit einer Rate von neuen Bildern pro Kanal alle 5 min zu erwerben. </li><li> Erwerben Sie die Bildsequenz mit der Übernahme-Protokoll oben eingegeben, indem Sie auf die &quot;Aufnahme / Aufnahme&quot; Taste. </li></ol> 5. Quantifizierung von Inclusion Wachstumsparameter in lebenden Zellen <ol><li> Zu gewünschten Zeitpunkten nach der Infektion (z. B. 24, 48 hpi) entfernen Chlamydia infiziert GFP-HeLa-Zellen von Inkubator und montieren auf einem invertierten Fluoreszenzmikroskop. Hinweis: A-20X oder 40X Klimaziel mit einer hohen NA ist am besten geeignet für 24-Well-Platten und ein 40X Öl-Objektiv ist für Kammer-Objektträgern empfohlen. Wachsen Zellen auf beiden Substrat für dieses assay. </li><li> Konzentrieren Sie sich auf Mittelebenen von Zellen, in denen Einschlüsse sind bei ihrer maximalen Durchmesser und liefern gestochen scharfe Kanten. </li><li> Erwerben Sie Bilder für viele Bereiche in jeder Vertiefung (mindestens 10 pro Loch); Brunnen stellen typischerweise Wiederholungen oder experimentelle Variablen. </li><li> Zählen Sie die Anzahl der Einschlüsse von Hand, mit dem Auge (Einschlüsse sind schwarz Fächer in GFP-HeLa-Zellen) oder verwenden Imaging-Software-Algorithmen, um automatisch zu identifizieren und zu zählen Einschlüsse. <ol><li> Finden GFP-HeLa-Zellen durch Fluoreszenzintensität (Befehl &#39;Objekte zu finden &quot;) und stellen Fluoreszenzschwellenintensität basierend auf relativen Intensitäten der Zellen. </li><li> Filter selektierten Objekte mit Größe Richtlinien halten diese nur größer als 5 &amp; mgr; m 2. Dies ist &#39;Bevölkerung 1&#39;. </li><li> Übernehmen &quot;Löcher füllen in Objects &#39;Befehl mit&quot; Bevölkerung 1&#39; als Eingang. Dieser Schritt erzeugt &quot;Bevölkerung 2 &#39;. </li><li> Subtract &quot;Bevölkerung 1&quot; von &quot;population 2 &#39;positiv geprägt Chlamydia Einschlüsse, bezeichnet als Ertrag &quot;Bevölkerung 3&#39;. </li><li> Bewerben Größe Filter auf (&#39;Filter Bevölkerung&#39; Kommando) &quot;Bevölkerung 3 &#39;, zum Beispiel halten Objekte größer als 25 &amp; mgr; m 2 für Einschlüsse in Zellen, die für 24 Stunden oder mehr infiziert. Für Frühinfektion Zeiten, wie vor 18 Stunden, stellen Größe Filter auf Objekte größer als 4 &amp; mgr; m 2 zu halten, da diese Einschlüsse sind viel kleiner. Größe Filterergebnisse in einer Population von als &quot;Einschlüsse&quot; bezeichneten Objekte. </li><li> Berechnen Sie quantitative Daten aus Bildern, zum Beispiel Einbeziehung Nummer, Aufnahme Größe und Einbindung Umfang, mit Imaging-Software. </li></ol></li></ol>

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology.">Cell biology.</a> Chlamydia: Intracellular Biology, Pathogenesis, and Immunity. , 101-138 (1999).</li><li>Wang, Y., Kahane, S., Cutcliffe, L. T., Skilton, R. J., Lambden, P. R., Clarke, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+transformation+system+for+Chlamydia+trachomatis:+restoration+of+glycogen+biosynthesis+by+acquisition+of+a+plasmid+shuttle+vector).">Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector).</a> PLoS Pathogens. 7 (9), e1002258 (2011).</li><li>Hackstadt, T., Scidmore, M. A., Rockey, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+metabolism+in+Chlamydia+trachomatis-infected+cells:+directed+trafficking+of+Golgi-derived+sphingolipids+to+the+chlamydial+inclusion.">Lipid metabolism in Chlamydia trachomatis-infected cells: directed trafficking of Golgi-derived sphingolipids to the chlamydial inclusion.</a> Proceedings of the National Academy of Sciences of the United States of America. 92 (11), 4877-4881 (1995).</li><li>Boleti, H., Ojcius, D. M., Dautry-Varsat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+labelling+of+intracellular+bacteria+in+living+host+cells.">Fluorescent labelling of intracellular bacteria in living host cells.</a> Journal of Microbiological Methods. 40 (3), 265-274 (2000).</li><li>Hybiske, K., Stephens, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+host+cell+exit+by+the+intracellular+bacterium+Chlamydia.">Mechanisms of host cell exit by the intracellular bacterium Chlamydia.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (27), 11430-11435 (2007).</li><li>Heinzen, R. A., Hackstadt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Chlamydia+trachomatis+parasitophorous+vacuolar+membrane+is+not+passively+permeable+to+low-molecular-weight+compounds.">The Chlamydia trachomatis parasitophorous vacuolar membrane is not passively permeable to low-molecular-weight compounds.</a> Infection and Immunity. 65 (3), 1088-1094 (1997).</li><li>Wickstrum, J., Sammons, L. R., Restivo, K. N., Hefty, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+expression+in+Chlamydia+trachomatis+using+the+tet+system.">Conditional gene expression in Chlamydia trachomatis using the tet system.</a> PLoS One. 8 (10), 10-1371 (2013).</li><li>Chin, E., Kirker, K., Zuck, M., James, G., Hybiske, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+recruitment+to+the+Chlamydia+inclusion+is+spatiotemporally+regulated+by+a+mechanism+that+requires+host+and+bacterial+factors.">Actin recruitment to the Chlamydia inclusion is spatiotemporally regulated by a mechanism that requires host and bacterial factors.</a> PLoS One. 7 (10), e46949 (2012).</li><li>Agaisse, H., Derr&#233;, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+C.+trachomatis+cloning+vector+and+the+generation+of+C.+trachomatis+strains+expressing+fluorescent+proteins+under+the+control+of+a+C.+trachomatis+promoter.">A C. trachomatis cloning vector and the generation of C. trachomatis strains expressing fluorescent proteins under the control of a C. trachomatis promoter.</a> PLoS One. 8 (2), e57090 (2013).</li></ol>

Die Autoren erklären, dass sie keine konkurrierenden finanziellen Interessen.

Hier beschreiben wir die experimentelle Strategie zur Erzeugung von Fluoreszenz-Wirtszellen für die Echtzeit-Visualisierung und Analyse von Chlamydia Einschlüsse. Diese Vakuole Visualisierung Ansatz eröffnet die leistungsfähige Möglichkeit, im Laufe der Zeit zu beleuchten, zu verfolgen und quantitativen Messung der dynamischen Eigenschaften von Chlamydieneinschlüsse in Populationen von Zellen oder in Einzelzellen. Chlamydia Einschlüsse in fluoreszierendem Protein markierte Zellen sind auffallend...

Infektionskrankheiten durch Arten der intrazelluläre Bakterium Chlamydia verursacht entlocken eine große Belastung für die globale Gesundheit, einschließlich sexuell übertragbare Krankheit, entzündliche Erkrankungen des Beckens, Blindheit, Pneumonie und möglicherweise Atherosklerose 1-4. Die Fähigkeit von Chlamydia um mit der Wirtszelle wechselwirken, innerhalb einer Vakuole (genannt die Aufnahme), ist ein kritischer Faktor für die erfolgreiche Infektion von Zellen und dem Host. Die Aufnahme ist eine neuartige pathogenen Fach, die Chlamydien-Wachstum ermöglicht und wird dynamisch über die gesamte 2-3 Tag Entwicklungszyklus von Chlamydia 5 modifiziert. Die obligat intrazelluläre Natur der Chlamydien stellt zahlreiche Herausforderungen für die Forschung, insbesondere für direkt an das Studium der einzigartigen Biologie der Eingliederung. Ein großes Handicap war die Unfähigkeit, entweder intrazellulären Chlamydien oder ihre Aufnahme von Fluoreszenz Ansatz effizient visualisierenes in lebenden Zellen. Eine neue Entdeckung hat endlich enthüllt die Mittel, um GFP-exprimierenden C. generieren trachomatis 6; aber dieses Ergebnis noch nicht auf eine spezifische Markierung der Aufnahme geführt. Einige Techniken zur Markierung von Bakterien und Einschlüsse 7,8 beschrieben worden, aber sie Mängel auf, wie nicht-Spezifität transiency und die Anfälligkeit gegen Ausbleichung leiden. Ein Schlüssel Entdeckung von unserer Arbeitsgruppe gegründet, eine neue Strategie für die Beleuchtung der Aufnahme mit GFP-exprimierenden Wirtszellen 9. Diese Strategie rationell nutzt die intrinsische Undurchlässigkeit der Einschlußmembran bis größer als 520 Da 10 Molekülen. Wenn Zellen entwickelt, um stabil eine bestimmte cytosolische fluoreszierende Protein (zB GFP oder mCherry) zum Ausdruck bringen, sind Chlamydia Einschlüsse mit außergewöhnlicher Klarheit durch ihre vollständigen Ausschluss der Fluoreszenz sichtbar. Diese umgekehrte Imaging-Strategie ermöglicht eine sofortige Visualisierung von Einschlüssen für alle Chlamydia Arten und es kann leicht für die meisten Wirtszellen von Interesse angepasst werden. Als Demonstration der Nützlichkeit wurde dieses Verfahren bisher verwendeten zu offenbaren und zu definieren, die zellulären Austrittswege für Chlamydia spp 9. Hier haben wir weiter zu zeigen, wie dieses Verfahren durchgeführt wird, und kann genutzt werden, um Schlüssel quantitative Daten über die Aufnahme Wachstumsdynamik abgeleitet werden. Darüber hinaus kann sie wirksam für teure Antikörper-basierte Zählverfahren ersetzen und kann in Kombination mit anderen fluoreszierenden Markern, wie mKate2 exprimierenden Chlamydia 11 verwendet werden. Diese leistungsstarke Kombination von Werkzeugen ermöglicht Erforschung der physikalischen Eigenschaften des Chlamydien-Aufnahme-Membran im Inneren lebenden Wirtszellen.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-Mem</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#181;m filters</td>
			<td>Fisher</td>
			<td>09-719D</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Invitrogen</td>
			<td>10131</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Invitrogen</td>
			<td>14025</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11995</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Invitrogen</td>
			<td>11875</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 w/o phenol red</td>
			<td>Cellgro</td>
			<td>17-105-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G</td>
			<td>Sigma</td>
			<td>13752</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>C7698</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Chamber slides, Lab-Tek II</td>
			<td>Nunc</td>
			<td>154526, 154534</td>
			<td></td>
		</tr>
	</tbody>
</table>

using fluorescent proteins to visualize and quantitate chlamydia vacuole growth dynamics in living cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Säugetierzellen, die cytosolische fluoreszierende Proteine ​​(zB GFP) kann konstruiert werden, um die Beleuchtung des Chlamydiaeinschlüssen einem lebenden, infizierten Zellkulturen zu ermöglichen. Nach Infektion mit Chlamydia, sind Einschlüsse leicht als schwarze Flecken in den Wirtszellen (Abbildung 1) sichtbar. Die Klarheit der fluoreszenz fehlt Einschlüsse für die automatisierte Identifikation der Einschlüsse in zahlreichen Bereichen und / oder den Behandlungen <...

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Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

Verwendung Fluoreszierende Proteine, zu visualisieren und zu quantifizieren<em&gt; Chlamydia</em&gt; Vakuole Wachstumsdynamik in lebenden Zellen

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

using-fluorescent-proteins-to-visualize-quantitate-chlamydia-vacuole

Research

JoVE Journal

Immunology and Infection

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

7.5K Aufrufe.  University of Washington. A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

Serie: Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Mess-Calpain Aktivität in fixierten und lebenden Zellen mittels Durchflusszytometrie

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Forschung

Immunologie und Infektion

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Seven Steps to Cells Stellate

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Nach Cell-Schicksal<em&gt; E. coli</em&gt; Nach Infektion mit Phagen

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. 
We generated model xenograft tumors in mice by subcutaneous injection of a human ovarian cancer cell line, OVCAR-8 (NCI DCTD Tumor Repository, Frederick, MD). 
We transformed attenuated strains of S. typhimurium bacteria (ELH430:SL1344 phoPQ- 1) with a constitutively expressed luciferase (luxCDABE) plasmid for visualization2. These strains specifically colonize tumors while remaining essentially non-virulent to the mouse1. 
Once measurable tumors were established, bacteria were injected intravenously via the tail vein with varying dosage. Tumor-localized, bacterial gene expression was monitored in real time over the course of 60 hours using an in vivo imaging system (IVIS). At each time point, tumors were excised, homogenized, and plated to quantitate bacterial colonies for correlation with gene expression data. 
Together, this data yields a quantitative measure of the in vivo growth and gene expression dynamics of bacteria growing inside tumors.

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. This video covers tumor cell preparation and implantation, bacteria preparation and injection, whole-animal luminescence imaging, tumor excision, and bacterial colony counting.

Mess-Wachstum und Genexpression von Tumor-Dynamics Targeted<em&gt; S. Typhimurium</em&gt; Bakterien

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium ...

Forward Genetic Approaches in Chlamydia trachomatis

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its intractability to experimental transformation with recombinant DNA. We developed an approach to perform genetic analysis in C. trachomatis&#160;despite the lack of molecular genetic tools. Our method involves: i.) chemical mutagenesis to rapidly generate comprehensive libraries of genetically-defined mutants with distinct phenotypes; ii.) whole-genome sequencing (WGS) to map the underlying genetic lesions and to find associations between mutated gene(s) and a common phenotype; iii.) generation of recombinant strains through co-infection of mammalian cells with mutant and wild type bacteria. Accordingly, we were able to establish causal relationships between genotypes and phenotypes. The coupling of chemically-induced gene variation and WGS to establish correlative genotype&#8211;phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.

Forward Genetic Approaches in Chlamydia trachomatis

We describe a methodology to perform genetic analysis in Chlamydia based on chemical mutagenesis and whole genome sequencing. In addition, a system for DNA exchange within infected cells is described that can be used for genetic mapping. This method may be broadly applicable to microbial systems lacking transformation systems and molecular genetic tools.

Vorwärts Genetische Ansätze in<em&gt; Chlamydia trachomatis</em

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Überwachen von Änderungen in Membran-Polarity, Membrane Integrity und intrazelluläre Ionenkonzentrationen in<em&gt; Streptococcus pneumoniae</em&gt; Verwenden von Fluoreszenzfarbstoffen

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Mit fluoreszierenden Proteinen zu Glycosome Dynamics in der afrikanischen Trypanosomen auf Monitor

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

&quot;Phagosome Closure Assay&quot; to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

&quot;Phagosomen Closure Assay&quot; zu visualisieren Phagosomen Formation in drei Dimensionen Mit Total Internal Reflection Fluoreszenz-Mikroskopie (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Precision-cut Maus Lung Slices Live-Pulmonary Dendritische Zellen sichtbar zu machen

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.