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. Generazione di linee cellulari Host fluorescente <ol><li> Giorno 1. Cellule piastra 293T (o altra linea di confezionamento retrovirali) su 6 pozzetti a 2 x 10 6 cellule / Beh, per essere ~ 75% confluenti il giorno successivo. Se lo si desidera, da utilizzare piastre pozzetti in duplicato per ciascuna retrovirus. </li><li> Giorno 2. Le cellule Aspirare e aggiungere 2 ml di terreno di coltura fresco (DMEM + 10% FBS + 2 mM L-glutammina). Trasfettare le cellule con 5-8 mcg ciascuna di vettore retrovirale (contenente GFP) e la confezione vettore (ad es., PVSVg) utilizzando Lipofectamine 2000 o reagente simile e seguendo le istruzioni del produttore. </li><li> Incubare le cellule con il DNA per 4-6 ore in una C CO 2 incubatore 37 °, e successivamente sostituire medio con 1 ml di terreno di coltura. </li><li> Incubare le cellule per 48 ore a 37 ° C CO 2 incubatore. </li><li> 3. Piatto cellule Day bersaglio (ad es., HeLa) su 6 pozzetti a una densità approssimativa di 10 5 cellule / pozzetto, per essere ~ 50% confluenti ilIl giorno dopo. Verificare trasfezione in cellule 293T positivo monitorando fluorescenza GFP su un microscopio invertito. </li><li> Giorno 4. Raccogliere-retrovirus contenente supernatanti le cellule 293T con una pipetta e filtrare il surnatante attraverso un filtro siringa da acetato di 0,45 micron di cellulosa. </li><li> Rimuovere medio da cellule HeLa e aggiungere 0,5 ml di mezzo di crescita, contenente 16 mg / ml polibrene. Aggiungi ~ 0,5 ml di retrovirus filtrato alle cellule goccia a goccia e incubare le cellule in una C CO 2 incubatore 37 ° per 24 ore. Conservare eventuali retrovirus rimanenti (cioè dal duplicare pozzo) a 4 ° C. </li><li> Giorno 5. Se sono stati fatti pozzetti in duplicato per generare retrovirus in più, ripetere il punto 1.7 di infettare in serie con le restanti particelle retrovirali. </li><li> Giorno 6. Passaggio transfettate cellule HeLa 1: 1 in 10 cm piatto contenente selezione antibiotica (ad esempio 500 mg / ml di neomicina). </li><li> Attendere un tempo sufficiente per cellule trasdotte a ricrescere in presenza di selezione antibiotic, dopo di che le cellule possono essere propagate per tecniche standard con una minore concentrazione di selezione antibiotica (es., 200 ug / ml di neomicina). Nota: Le cellule possono anche essere selezionati per clonale luminosità ideale in questo momento, se necessario. </li></ol> 2. Generazione di GFP che esprimono Chlamydia trachomatis <ol><li> Preparare 2x CaCl 2 tampone (20 mM Tris pH 7,4, 100 mM CaCl 2) e tampone 4SP (0,4 M di saccarosio, 16 mM Na 2 HPO 4). </li><li> Giorno 1. Scongelare congelato C. trachomatis magazzino dal ghiaccio e portare subito 10 microlitri di batteri in 50 microlitri 2x CaCl buffer di 2. Aggiungere 3 mg plasmide DNA (ad es., Pask-GFP-mKate2-L2), mescolare bene e incubare per 30 minuti a 25 ° C. </li><li> Durante questa fase, preparare cellule ospiti da trypsinizing un pallone T-75 di cellule McCoy in una provetta da centrifuga. Centrifugare la sospensione cellulare a 50 xg per 5 minuti e scartare il surnatante. Resuspend pellet cellulare in volume appropriato di 1x tampone CaCl 2 per ottenere una concentrazione finale di 4 x 10 7 cellule / ml. </li><li> Dopo 30 min di incubazione (dal punto 2.2), aggiungere 100 ml preparati cellule McCoy per tubo miscela trasformazione (volume totale 200 ml) ed incubare un ulteriore 20 min a 25 ° C. Aggiungere 100 ml di questa miscela cellula trasformata per un singolo bene da un 6-pozzetti e overlay con mezzo di 2 ml di crescita. Incubare a 37 ° C in un incubatore CO 2 per 2 giorni. </li><li> Giorno 3. Le cellule Lyse McCoy infettate con C. trachomatis sostituendo medio con 1 ml di dH 2 O e raschiare le cellule con un piegato punta pipetta 1000 ml. Aggiungere 1 ml di tampone 4SP e passare sospensione attraverso un ago 27,5 G ~ 5 volte per lisi cellulare efficiente e il rilascio di C. trachomatis. </li><li> Trasferimento 2 ml di lisato cellulare contenente C. trachomatis in un pallone T-75 contenente un monostrato confluente di fresco placcato McCcellule Oy; aggiungere 8 ml DMEM (senza FBS) e incubare per 2 ore a 25 ° C per consentire C. trachomatis di aderire alle cellule. </li><li> Aspirare le cellule e aggiungere terreno di coltura fresco supplementato con 10 U / ml di penicillina G e 1 mg / ml cicloeximide. Incubare per 48 h beuta in un incubatore a 37 ° C CO 2. </li><li> Giorno 4. Verificare al microscopio ottico che le cellule sono infettate e inclusioni contengono Chlamydia aberrante. Identificare inclusioni come vacuoli luminosi su un microscopio a luce normale, e corpi clamidia aberranti come oggetti anulari all&#39;interno di inclusioni che sono maggiori di 2 micron di diametro. </li><li> Giorno 5. Lyse cultura sostituendo medie, con 2 ml di dH 2 O e raschiare le cellule in una sospensione. Aggiungere 2 ml di tampone 4SP e passare sospensione attraverso un ago 27,5 G ~ 5 volte. </li><li> Utilizzare 2 ml di lisato cellulare per infettare un nuovo monostrato di cellule McCoy in un singolo pozzetto in una piastra da 6 pozzetti. Incubare le cellule con lisato per 2 ore a 25 ° C, aspirare e aggiungere la crescita frescoterreno contenente penicillina G e cicloesimide. </li><li> Incubare le cellule in un incubatore a 37 ° C CO 2 per 3-5 giorni, a seconda di salute generale delle cellule. </li><li> Ripetere i punti 2.9-2.10 una o più volte, se necessario, le culture passaging nei pozzetti fresche in un 6-bene fino a quando le normali inclusioni ricerca (privi di organi aberranti) sono emerse. In questo momento utilizzare un microscopio a fluorescenza invertito per controllare che C. trachomatis esprimere mKate2 (rosso). </li><li> Scalare colture infette per la produzione delle scorte di clamidia ad alto titolo, ad esempio raccogliendo trasformanti Chlamydia da cellule infettate McCoy o HeLa cresciute in T-150 palloni. </li></ol> 3. infettare le cellule con Chlamydia <ol><li> Piastra cellule GFP-HeLa sulla nave cultura desiderato, ad esempio piatti di vetro inferiore o diapositive da camera per imaging ad alta risoluzione, o piastre da 24 pozzetti per la determinazione IFU e lo screening pozzetti. </li><li> Scongelare frotubo zen contenente C. trachomatis, C. muridarum, o C. pneumoniae su ghiaccio e diluire in HBSS alla molteplicità desiderata di infezione (MOI), per esempio MOI = 1. </li><li> Eseguire infezioni statiche o centrifugazione-aided base al particolare ceppo Chlamydia che verrà utilizzato. <ol><li> Per le infezioni da C. trachomatis LGV sierotipo L2 o C. muridarum, lavare le cellule GFP-HeLa con HBSS e incubare con i batteri diluito per 2 ore a 25 ° C. Aspirare e lavare le cellule con HBSS, e incubare cellule in terreno di coltura a 37 ° C CO 2 incubatore. </li><li> Per le infezioni da C. trachomatis sierotipo D o C. pneumoniae, lavare le cellule GFP-HeLa con HBSS e aggiungere batteri diluiti alle cellule. Mettere piatto pozzetti o un piatto fondo di vetro (fissato con nastro in un coperchio piatto pozzetti) in un supporto di pozzetti piatto di un rotore attacco oscillante secchio in una centrifuga da banco. </li><li> Centrifugare le cellule a 900 xg a 25 ° C per 1 ora. </li&gt; <li> Rimuovere recipienti di coltura da centrifuga e posto in un incubatore a 37 ° C di CO 2 per 1 ora. </li><li> Aspirare le cellule in un armadio biosicurezza, lavare le cellule due volte con HBSS, e aggiungere mezzo di crescita fresco. Cellule posto in un incubatore CO 2 C 37 °. </li></ol></li></ol> 4. Visualizzazione cellulare dal vivo di Chlamydia Inclusioni <ol><li> Rimuovere Chlamydia infettate cellule GFP-HeLa di CO 2 incubatore e sostituirlo media con RPMI senza rosso fenolo + 5% FBS. </li><li> Montare le cellule su un microscopio a fluorescenza invertito (Nikon Eclipse Ti-E o simile) con un obiettivo 20X o superiore olio. </li><li> Utilizzando il software di imaging (Volocity 6 o simile), si concentrerà su e identificare Chlamydia infettato cellule GFP-HeLa: cellule verdi contenenti grandi buchi neri (inclusioni Chlamydia). </li><li> Segnare le posizioni xyz delle regioni contenenti Chlamydia cellule -infected di interesse utilizzando software di imaging (Volocity 6 o simile). Eseguire questo manualmente &#39;Aggiunta di punti &quot;, mentre nella modalità di visualizzazione XY Stage. In questo modo, le coordinate xyz vengono salvate per ogni punto contrassegnato. </li><li> Nella finestra di dialogo Installazione di acquisizione, configurare il software per l&#39;acquisizione verde (GFP, HeLa citoplasma) e rosso (mKate2, C. trachomatis) canali, e ad un tasso di nuovi fotogrammi al canale ogni 5 min. </li><li> Acquisire la sequenza di immagini tramite l&#39;acquisizione protocollo è entrato sopra cliccando sul pulsante &quot;Capture / Record&quot;. </li></ol> 5. La quantificazione dei parametri di crescita di inclusione in cellule vive <ol><li> A volte desiderato dopo l&#39;infezione (ad es., 24, 48 HPI) rimuovere Chlamydia infettato cellule GFP-HeLa da incubatore e montare su un microscopio a fluorescenza invertito. Nota: Un obiettivo 20X o 40X aria con un alto NA è più adatto per piastre da 24 pozzetti, e un obiettivo di olio 40X è raccomandato per le diapositive da camera. Crescere le cellule su entrambi substrato per questo culoay. </li><li> Focus su midplane di cellule, in cui inclusioni sono al loro diametro massimo e produrre bordi nitidi. </li><li> Acquisire le immagini per molti campi a ciascun pozzetto (almeno il 10 per bene); pozzi in genere rappresentano repliche o variabili sperimentali. </li><li> Enumerare il numero di inclusioni manualmente, a occhio (inclusioni sono compartimenti neri nelle cellule GFP-HeLa) o utilizzare algoritmi software di imaging per identificare e contare automaticamente inclusioni. <ol><li> Trova le cellule GFP-HeLa di intensità di fluorescenza (&#39;Trova oggetti&#39; di comando) e regolare l&#39;intensità soglia di fluorescenza in base a intensità relative delle cellule. </li><li> Filtrare gli oggetti selezionati con le linee guida di dimensioni di mantenere solo quelli maggiori di 5 micron 2. Si tratta di &#39;popolazione 1&#39;. </li><li> Applica &#39;riempire i fori in Objects comando utilizzando&#39; popolazione 1 &#39;come ingresso. Questo passaggio genera &#39;popolazione 2&#39;. </li><li> Sottrarre &#39;popolazione 1&#39; da &#39;pOPOLAZIONE 2 &#39;a cedere positivamente segnato inclusioni Chlamydia, designati come&#39; popolazione 3 &#39;. </li><li> Applicare il filtro dimensione di &#39;popolazione 3&#39; (comando &#39;Filter Popolazione&#39;), ad esempio, mantenere gli oggetti superiore a 25 micron 2 per inclusioni in cellule infettate per 24 ore o più a lungo. Per i tempi di infezione precoce, come ad esempio prima di 18 ore, filtrare dimensioni impostate per tenere gli oggetti maggiori di 4 micron 2, come queste inclusioni sono molto più piccoli. Risultati Dimensione filtro in una popolazione di oggetti designati come &quot;inclusioni&quot;. </li><li> Calcolare i dati quantitativi dalle immagini, ad esempio, il numero di inclusione, la dimensione di inclusione, e la circonferenza inclusione, utilizzando software di imaging. </li></ol></li></ol>

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	<li>Stamm, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlamydia+trachomatis+infections:+progress+and+problems.">Chlamydia trachomatis infections: progress and problems.</a> The Journal of Infectious Diseases. 179, S380-S383 (1999).</li><li>Schachter, J., 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=Infection+and+disease+epidemiology.">Infection and disease epidemiology.</a> Chlamydia: Intracellular Biology, Pathogenesis, and Immunity. , 139-169 (1999).</li><li>Campbell, L. A., Kuo, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlamydia+pneumoniae--an+infectious+risk+factor+for+atherosclerosis.">Chlamydia pneumoniae--an infectious risk factor for atherosclerosis.</a> Nature Reviews Microbiology. 2 (1), 23-32 (2004).</li><li>Darville, T., Hiltke, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+genital+tract+disease+due+to+Chlamydia+trachomatis.">Pathogenesis of genital tract disease due to Chlamydia trachomatis.</a> The Journal of Infectious Diseases. 201, S114-S125 (2010).</li><li>Hackstadt, T., 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=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>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Qui si descrive la strategia sperimentale per la generazione di cellule ospiti fluorescenti per la visualizzazione in tempo reale e l&#39;analisi delle inclusioni Chlamydia. Questo approccio visualizzazione vacuolo conferisce il potente capacità di illuminare, monitorare e quantitativamente misurare le proprietà dinamiche delle inclusioni clamidia tra popolazioni di cellule o in singole cellule nel corso del tempo. Inclusioni Chlamydia nelle cellule proteina marcata fluorescenti sono sorprendentement...

Le malattie infettive causate da specie del batterio Chlamydia intracellulare suscitare un peso importante sulla salute globale, tra cui malattia sessualmente trasmessa, malattia infiammatoria pelvica, la cecità, la polmonite e, eventualmente, l&#39;aterosclerosi 1-4. La capacità di Chlamydia di interagire con la cellula ospite, all&#39;interno di un vacuolo (chiamato l&#39;inclusione), è un fattore determinante per il loro successo infezione delle cellule e l&#39;host. L&#39;inclusione è un compartimento patogeno romanzo che permette la crescita da Chlamydia e viene modificato dinamicamente durante l&#39;intero ciclo di sviluppo di 2-3 giorni di Chlamydia 5. La natura obbligato intracellulare di chlamydiae presenta numerose sfide per la comunità di ricerca, in particolare per lo studio della biologia direttamente unico dell&#39;inclusione. Un importante ostacolo è stata l&#39;impossibilità di visualizzare in modo efficiente o Chlamydia intracellulare o la loro inclusione con approccio fluorescentees nelle cellule viventi. Una recente scoperta ha finalmente rivelato i mezzi per generare GFP che esprimono C. trachomatis 6; tuttavia, questo risultato non ha ancora portato a un&#39;etichettatura specifica dell&#39;inclusione. Alcune tecniche sono state descritte per l&#39;etichettatura di batteri e inclusioni 7,8, ma soffrono di carenze quali la non specificità, transitorietà e la suscettibilità alle photobleaching. Una scoperta fondamentale per il nostro gruppo ha stabilito una nuova strategia per illuminare l&#39;inclusione mediante GFP che esprimono cellule ospiti 9. Questa strategia sfrutta razionalmente l&#39;impermeabilità intrinseca della membrana inclusione di molecole maggiore di 520 Da 10. Quando le cellule sono progettati per esprimere stabilmente una particolare proteina fluorescente citosolica (ad esempio, GFP o mCherry), inclusioni Chlamydia sono visibili con notevole chiarezza la loro completa esclusione di fluorescenza. Questa strategia di imaging inversa permette la visualizzazione immediata di inclusioni per tutti Chlspecie amydia e può essere facilmente adattato per la maggior parte delle cellule ospiti d&#39;interesse. A dimostrazione della sua utilità, questo metodo è stato utilizzato in precedenza per rivelare e definire le vie di uscita per Chlamydia spp cellulari 9. Qui, abbiamo ulteriormente dimostra come si esegue questo metodo, e può essere sfruttata per ricavare dati quantitativi chiave su dinamiche di crescita inclusione. Inoltre, si può effettivamente sostituire costosi metodi di enumerazione base di anticorpi e può essere utilizzato in tandem con altre etichette fluorescenti, come mKate2 esprimono Chlamydia 11. Questa potente combinazione di strumenti consente l&#39;esplorazione delle proprietà fisiche della membrana inclusione chlamydial all&#39;interno delle cellule ospiti vivente.

<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.

Le cellule di mammiferi che esprimono proteine ​​fluorescenti citosoliche (ad esempio, GFP) possono essere progettati per consentire l&#39;illuminazione di inclusioni Chlamydia nelle colture cellulari, vivi infetti. Al momento l&#39;infezione da Chlamydia, inclusioni sono facilmente visibili come macchie nere nelle cellule ospiti (Figura 1). La chiarezza di inclusioni-fluorescenza carente può essere sfruttata per l&#39;identificazione automatizzata di inclusioni in numero...

Watch this Scientific Journal Video about Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells at JoVE.com

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.

Utilizzo di proteine ​​fluorescenti per visualizzare e Quantificazione<em&gt; Chlamydia</em&gt; Vacuolo crescita dinamica in cellule viventi

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 Views.  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.

Video: 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.

Misurazione dell&#39;attività Calpain nelle celle fissi e Living in citometria a flusso

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

Ricerca

Immunologia e infezioni

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.

Sette passi per Stellate Cellule

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.

A seguito di Cell-destino<em&gt; E. coli</em&gt; Dopo l&#39;infezione da fago 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 ...

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.

Crescita di misura e di espressione genica Dinamica di tumore-mirata<em&gt; S. Typhimurium</em&gt; Batteri

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.

Approcci genetici in avanti<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.

Monitoraggio dei cambiamenti di polarità membrana, membrana integrità e intracellulare di ioni concentrazioni in<em&gt; Streptococcus pneumoniae</em&gt; Utilizzo di tinture fluorescenti

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.

Utilizzo di proteine ​​fluorescenti per monitorare Glycosome Dynamics in Tripanosoma africano

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;Phagosome chiusura Assay&quot; per visualizzare phagosome Formazione in tre dimensioni utilizzando la riflessione totale interna fluorescente Microscopy (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.

Taglio di precisione polmone mouse Fette per la visualizzazione in diretta cellule dendritiche polmonare

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.

Utilizzo di proteine ​​fluorescenti per visualizzare e Quantificazione

Utilizzo di proteine fluorescenti per visualizzare e Quantificazione