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. Génération de lignées cellulaires hôtes Fluorescent <ol><li> Jour 1. Plate cellules 293T (ou autre ligne d&#39;emballage retroviral) sur des plaques à 6 puits à 2 x 10 6 cellules / puits, à ~ 75% de confluence le lendemain. Si on le souhaite, la plaque de puits en double pour chaque rétrovirus à être utilisés. </li><li> Jour 2. Aspirer cellules et ajouter 2 ml de milieu de croissance frais (DMEM + 10% de FBS + 2 mM de L-glutamine). Transfecter des cellules avec 5-8 ug de chaque vecteur retroviral (contenant la GFP) et le vecteur de l&#39;emballage (par ex., PVSVg) en utilisant de la Lipofectamine 2000 ou similaire réactif et en suivant les instructions du fabricant. </li><li> Incuber les cellules avec de l&#39;ADN de 4-6 heures dans un incubateur à CO 2 C 37 ° C, et ensuite remplacer milieu avec 1 ml de milieu de croissance. </li><li> Incuber les cellules pendant 48 heures à 37 ° C CO 2 incubateur. </li><li> Jour 3. cellules de plaques cibles (par ex., HeLa) sur des plaques à 6 puits à une densité approximative de 10 5 cellules / puits, à ~ 50% de confluence lele prochain jour. Vérifier positif transfection dans des cellules 293T en surveillant fluorescence de la GFP sur un microscope inversé. </li><li> Jour 4. Recueillir surnageants hors des cellules 293T aide d&#39;une pipette et filtrer le surnageant à travers un filtre d&#39;acétate de la seringue de 0,45 um de cellulose contenant un rétrovirus. </li><li> Retirer moyen de cellules HeLa et ajouter du milieu 0,5 ml de croissance contenant 16 pg / ml de polybrène. Ajouter ~ 0,5 ml de rétrovirus filtrée aux cellules goutte à goutte et incuber les cellules dans un C CO 2 incubateur à 37 ° pendant 24 heures. Rangez les éventuels rétrovirus restants (à savoir, à partir du puits en double) à 4 ° C. </li><li> Jour 5. Si les puits en double ont été effectués afin de générer rétrovirus supplémentaire, répétez l&#39;étape 1.7 pour infecter en série avec les particules rétrovirales restants. </li><li> Jour 6. Passage transfectées cellules HeLa 1: 1 dans une boîte de 10 cm contenant le choix des antibiotiques (par exemple, 500 ug / ml de néomycine). </li><li> Attendez suffisamment de temps pour les cellules transduites à repousser en présence de sélection antibiotic, après quoi les cellules peuvent être propagées par des techniques standard avec une concentration plus faible de sélection antibiotique (par ex., 200 pg / ml de néomycine). Note: Les cellules peuvent également être choisis par clonage pour la luminosité idéale à ce moment, si nécessaire. </li></ol> 2. Génération de la GFP exprimant Chlamydia trachomatis <ol><li> Préparer 2x CaCl2 tampon (20 mM Tris pH 7,4, 100 mM de CaCl2) et le tampon 4SP (0,4 M de saccharose, 16 mM de Na 2 HPO 4). </li><li> Jour 1. Décongelez C. trachomatis de stock sur la glace et diluer immédiatement 10 bactéries ul dans 50 pl 2x tampon CaCl 2. Ajouter 3 ADN plasmidique pg (par ex., PASK-GFP-mKate2-L2), bien mélanger et incuber pendant 30 min à 25 ° C. </li><li> Au cours de cette étape, la préparation des cellules hôtes par trypsinisation un flacon T-75 de cellules de McCoy dans un tube de centrifugeuse. Centrifuger la suspension cellulaire à 50 g pendant 5 min et jeter le surnageant. Resuspend culot cellulaire en volume approprié de tampon de CaCl2 1x pour donner une concentration finale de 4 x 10 7 cellules / ml. </li><li> Après l&#39;incubation de 30 minutes (de l&#39;étape 2.2), ajouter 100 ul de cellules de McCoy préparés tube de mélange de transformation (volume total 200 ul) et incuber 20 minutes supplémentaires à 25 ° C. Ajouter 100 ul de ce mélange de cellules transformées à un seul puits d&#39;une plaque à 6 puits avec du milieu de recouvrement et 2 ml de la croissance. Incuber à 37 ° C dans un incubateur à CO 2 pendant 2 jours. </li><li> Jour 3. Lyse cellules McCoy infectées par le C. trachomatis en remplaçant moyenne avec 1 ml dH 2 O et le grattage des cellules avec une pipette de 1000 pi plié. Ajouter 1 ml de tampon 4SP et passer la suspension à travers une aiguille de 27,5 G ~ 5 fois pour la lyse cellulaire efficace et la libération de C. trachomatis. </li><li> Transfer 2 ml de lysat cellulaire contenant C. trachomatis dans un flacon T-75 contenant une monocouche confluente de fraîchement plaqué McCcellules Oy; ajouter 8 ml de DMEM (sans FBS) et incuber pendant 2 heures à 25 ° C pour permettre C. trachomatis à adhérer aux cellules. </li><li> Aspirer les cellules et ajouter milieu de croissance frais supplémenté avec 10 U / ml de pénicilline G et 1 ug / ml de cycloheximide. Incuber pendant 48 h ballon dans un incubateur à 37 ° C de CO 2. </li><li> Jour 4. Vérifiez par microscopie optique que les cellules sont infectées et les inclusions contiennent Chlamydia aberrante. Identifier les inclusions que vacuoles lumineuses sur un microscope optique standard, et les organismes de Chlamydia aberrantes comme des objets en forme d&#39;anneau dans inclusions qui sont supérieures à 2 pm de diamètre. </li><li> Jour 5. Lyse culture en remplaçant moyenne avec 2 ml dH2Û et gratter les cellules en suspension. Ajouter 2 ml de tampon 4SP et passer la suspension à travers une aiguille de 27,5 G ~ 5 fois. </li><li> Utiliser 2 ml de lysat cellulaire à infecter une monocouche fraîche de cellules de McCoy dans un seul puits dans une plaque à 6 puits. Incuber les cellules avec lysat pendant 2 heures à 25 ° C, aspirer et ajouter croissance fraismilieu contenant de la pénicilline G et cycloheximide. </li><li> Incuber les cellules dans un incubateur à 37 ° C de CO 2 pendant 3-5 jours, en fonction de la santé générale des cellules. </li><li> Répétez les étapes 2.9-2.10 une ou plusieurs fois, selon les besoins, les cultures des passages dans des puits frais dans une plaque à 6 puits jusqu&#39;à ce que les inclusions d&#39;apparence normale (dépourvus de corps aberrantes) ont vu le jour. A ce moment utiliser un microscope inversé à fluorescence pour vérifier que C. trachomatis exprimer mKate2 (rouge). </li><li> Intensifier les cultures infectées pour la production des stocks de Chlamydia de titre élevé, par exemple en récoltant transformants Chlamydia à partir de cellules McCoy ou HeLa infectées cultivées en flacons T-150. </li></ol> 3. infection de cellules avec Chlamydia <ol><li> Plate cellules HeLa GFP-sur récipient de culture souhaitée, par exemple des plats de fond de verre ou des diapositives de la chambre pour l&#39;imagerie haute résolution, ou des plaques à 24 puits pour la détermination IFU et le dépistage à puits multiples. </li><li> Décongeler froTube zen contenant C. trachomatis, C. muridarum, ou C. pneumoniae sur de la glace et diluées dans du HBSS à la multiplicité désirée de l&#39;infection (MOI), par exemple MOI = 1. </li><li> Effectuer infections statiques ou assistée par centrifugation en fonction de la souche de Chlamydia particulier qui sera utilisé. <ol><li> Pour les infections à C. trachomatis LGV sérotype L2 ou C. muridarum, laver les cellules GFP-HeLa avec HBSS et incuber des bactéries dilué pendant 2 heures à 25 ° C. Aspirer et laver les cellules avec HBSS, et incuber les cellules dans un milieu de croissance de 37 ° C CO 2 incubateur. </li><li> Pour les infections à C. trachomatis serotype D ou C. pneumoniae, laver les cellules GFP-HeLa avec HBSS et ajouter des bactéries aux cellules dilué. Placez plaque multi-puits ou un plat à fond de verre (fixé par un ruban dans un couvercle de plaque multi-puits) dans un support de plaque à puits multiples d&#39;un rotor attachement à godet oscillant dans une centrifugeuse de paillasse. </li><li> Centrifuger les cellules à 900 g à 25 ° C pendant 1 h. </li&gt; <li> Retirer les contenants de la culture d&#39;une centrifugeuse et placer dans un incubateur à 37 ° C CO 2 pendant 1 heure. </li><li> Aspirer cellules dans une enceinte de sécurité biologique, laver les cellules deux fois avec HBSS, et ajouter du milieu de croissance frais. La place des cellules dans un C CO 2 incubateur à 37 °. </li></ol></li></ol> 4. cellules vivantes Visualisation de Chlamydia Inclusions <ol><li> Retirer Chlamydia infectés cellules GFP-HeLa de CO 2 incubateur et remplacer moyenne avec RPMI sans rouge de phénol + 5% de FBS. </li><li> Cellules de montage sur un microscope à fluorescence inversé (Nikon Eclipse Ti-E ou similaire) en utilisant un objectif 20X ou plus d&#39;huile. </li><li> En utilisant un logiciel d&#39;imagerie (Volocity 6 ou similaire), se concentrer sur et identifier les cellules infectées par Chlamydia GFP-HeLa: cellules vertes contenant de grandes trous noirs (inclusions de Chlamydia). </li><li> Marquer les endroits xyz de régions contenant des cellules infectées par Chlamydia d&#39;intérêt en utilisant un logiciel d&#39;imagerie (Volocity 6 ou similaire). Effectuez cette manuellement par «Ajout de points de la tandis que dans le mode d&#39;affichage XY scène. De cette manière, coordonnées XYZ sont enregistrées pour chaque emplacement marqué. </li><li> Dans la boîte de dialogue de configuration d&#39;acquisition, configurer le logiciel pour acquérir verte (GFP, HeLa cytosol) et rouge (mKate2, C. trachomatis) chaînes, et à un taux de nouvelles images par canal toutes les 5 min. </li><li> Acquérir la séquence d&#39;images en utilisant l&#39;acquisition protocole est entré ci-dessus en cliquant sur le &quot;Capture / Enregistrement&quot; bouton. </li></ol> 5. La quantification des paramètres de croissance d&#39;inclusion dans les cellules vivantes <ol><li> À certains moments souhaités post-infection (par ex., 24, 48 HPI) éliminer les cellules infectées par Chlamydia GFP-HeLa de l&#39;incubateur et monter sur un microscope à fluorescence inversé. Note: Un objectif 20X ou 40X air avec une haute NA est le mieux adapté pour les plaques à 24 puits, et un objectif de l&#39;huile 40X est recommandé pour les diapositives de la chambre. Cultiver les cellules de chaque substrat pour cet âneAy. </li><li> Focus sur plan médian de cellules, où les inclusions sont à leur diamètre maximum et obtenir des bords nets. </li><li> Acquérir les images pour les nombreux domaines dans chaque puits (au moins 10 par puits); puits représentent généralement les répliques ou les variables expérimentales. </li><li> Énumérer le nombre d&#39;inclusions manuellement, par l&#39;oeil (les inclusions sont compartiments noirs dans les cellules HeLa-GFP) ou utiliser des algorithmes de logiciels d&#39;imagerie pour identifier automatiquement et compter inclusions. <ol><li> Trouver cellules GFP-HeLa par l&#39;intensité de fluorescence («Trouvez les objets de la commande) et d&#39;ajuster l&#39;intensité de seuil de fluorescence en fonction des intensités relatives des cellules. </li><li> Filtrer les objets sélectionnés à l&#39;aide des lignes directrices de la taille de garder ceux-là seulement supérieure à 5 pm 2. Ceci est «population 1 &#39;. </li><li> Appliquer &quot;remplir les trous dans les objets de la commande à l&#39;aide de« population 1 &#39;comme entrée. Cette étape génère «population 2 &#39;. </li><li> «Population 1 &#39;Soustraire de&#39; population 2 &#39;pour donner positivement marqué inclusions de Chlamydia, désignés comme «population 3&#39;. </li><li> Appliquer un filtre de taille pour &#39;population 3&#39; (&#39;Filtrez population&#39; commande), par exemple des objets de maintien supérieure à 25 um 2 pour les inclusions dans les cellules infectées pendant 24 heures ou plus. Pour des temps de début de l&#39;infection, comme avant 18 heures, réglez filtre de taille pour garder les objets de plus de 4 pm 2, que ces inclusions sont beaucoup plus petits. Les résultats de filtrage de taille dans une population d&#39;objets désignés comme des «inclusions». </li><li> Calculer des données à partir d&#39;images quantitative, par exemple, le numéro de l&#39;inclusion, la taille de l&#39;inclusion, et l&#39;inclusion circonférence, en utilisant un logiciel d&#39;imagerie. </li></ol></li></ol>

<ol>
	<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>

Les auteurs déclarent qu&#39;ils ont aucun intérêt financier concurrentes.

Nous décrivons ici la stratégie expérimentale pour générer des cellules hôtes fluorescents pour de vrai visualisation et l&#39;analyse des inclusions de Chlamydia temps. Cette approche vacuole de visualisation confère la capacité puissante pour éclairer, de suivre et de mesurer quantitativement les propriétés dynamiques des inclusions à Chlamydia dans les populations de cellules ou dans des cellules individuelles au fil du temps. Inclusions de Chlamydia dans les cellules de protéine marqu?...

Les maladies infectieuses causées par des espèces de la bactérie Chlamydia intracellulaire suscitent un fardeau important sur ​​la santé mondiale, y compris les maladies sexuellement transmissibles, la maladie inflammatoire pelvienne, la cécité, la pneumonie et, éventuellement, l&#39;athérosclérose 1-4. La capacité de Chlamydia d&#39;interagir avec la cellule hôte, à partir de l&#39;intérieur d&#39;une vacuole (appelée inclusion), est un facteur déterminant de leur infection réussie de cellules et de l&#39;hôte. L&#39;inclusion est un compartiment pathogène roman qui permet la croissance de la chlamydia et est modifié dynamiquement tout au long du cycle de développement 2-3 jours de Chlamydia 5. La nature intracellulaire obligatoire de Chlamydia présente de nombreux défis à la communauté de la recherche, en particulier pour étudier directement la biologie unique de l&#39;inclusion. Un handicap majeur a été l&#39;incapacité de visualiser efficacement soit Chlamydia intracellulaire ou leur inclusion par l&#39;approche fluorescentees dans les cellules vivantes. Une découverte récente a finalement révélé les moyens de générer exprimant la GFP C. 6 trachomatis; Toutefois, cette constatation n&#39;a pas encore abouti à un étiquetage spécifique de l&#39;inclusion. Certaines techniques ont été décrites pour l&#39;étiquetage des bactéries et des inclusions 7,8, mais ils souffrent de lacunes tels que la non-spécificité, l&#39;éphémère et la susceptibilité à photoblanchiment. Une découverte clé de notre groupe a établi une nouvelle stratégie pour éclairer l&#39;inclusion en utilisant exprimant la GFP cellules hôtes 9. Cette stratégie exploite rationnellement l&#39;imperméabilité intrinsèque de la membrane de l&#39;inclusion de molécules supérieur à 10 520 Da. Lorsque les cellules sont manipulées pour exprimer de façon stable une protéine fluorescente cytosolique particulier (par exemple, la GFP ou mCherry), inclusions à Chlamydia sont visibles avec une clarté remarquable par leur exclusion complète de la fluorescence. Cette stratégie d&#39;imagerie inverse permet la visualisation immédiate des inclusions pour tous Chlespèces amydia et il peut être facilement adaptés pour la plupart des cellules hôtes d&#39;intérêt. Comme une démonstration de son utilité, cette méthode a été utilisée précédemment pour révéler et de définir les voies de sortie cellulaires pour Chlamydia spp 9. Ici, nous démontrons encore comment cette méthode est effectuée, et peut être exploitée pour obtenir des données quantitatives clés sur la dynamique de croissance de l&#39;inclusion. En outre, il peut effectivement se substituer aux méthodes de dénombrement à base d&#39;anticorps coûteux et peut être utilisé en tandem avec d&#39;autres marqueurs fluorescents, comme mKate2 exprimant Chlamydia 11. Cette puissante combinaison d&#39;outils permet l&#39;exploration des propriétés physiques de la membrane de l&#39;inclusion Chlamydia intérieur des cellules hôtes de vie.

<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.

Les cellules de mammifères exprimant des protéines cytosoliques fluorescents (par exemple, GFP) peuvent être conçus pour permettre un éclairage d&#39;inclusions à Chlamydia, dans des cultures de cellules infectées vivantes. Lors de l&#39;infection à Chlamydia, les inclusions sont facilement visibles sous forme de taches noires dans les cellules hôtes (figure 1). La clarté des inclusions de fluorescence manque peut être exploitée pour l&#39;identification automatiqu...

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.

Utilisation de protéines fluorescentes de visualiser et de quantifier<em&gt; Chlamydia</em&gt; Vacuole la dynamique de croissance dans les cellules vivantes

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 vues.  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.

Vidéo: 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.

Mesure de l&#39;activité calpaïne dans des cellules fixées et de la Vie par cytométrie en flux

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

Recherche

Immunologie et infection

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.

Sept étapes pour Stellate Cellules

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.

Après Cell-sort<em&gt; E. coli</em&gt; Après infection par le 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 ...

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.

Mesurer la croissance et Gene Expression Dynamique de tumeur ciblée<em&gt; S. Typhimurium</em&gt; Bacteria

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.

Approches avant génétiques dans<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.

La surveillance des changements dans la membrane de polarité, Membrane intégrité, et intracellulaires d&#39;ions concentrations dans<em&gt; Streptococcus pneumoniae</em&gt; Utilisation des colorants fluorescents

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.

Utilisation de protéines fluorescentes pour surveiller glycosome Dynamics dans le trypanosome africain

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 Clôture Assay&quot; pour visualiser Formation Phagosome en trois dimensions en utilisant la réflexion interne totale Fluorescent 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.

coupe de précision de la souris Tranches Lung pour visualiser les cellules vivantes pulmonaire dendritiques

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.