Cette recherche a été financée en partie par NIH prix 5T32AI07506, 5R01AI033774, 5R37AI033142, 5R01AI052733.

Tous les travaux de l&#39;animal a été fait en conformité avec les règlements et les lignes directrices de l&#39;Institut d&#39;études animales de l&#39;Albert Einstein College of Medicine. 1. Conditions de croissance de C. neoformans H99 (sérotype A) <ol><li> Croissance individuelle C. neoformans (Cn) colonies sur des plaques de gélose Sabouraud. </li><li> Un jour avant l&#39;expérience, sélectionnez une colonie et inoculer 10 ml de bouillon de Sabouraud. </li><li> Laisser la culture se développer pendant une nuit à 37 ° C sous agitation à une vitesse de 250 tours par minute. </li></ol> 2 Isolement et préparation des os primaire macrophages de C57 / BL6 souris <ol><li> Euthanasier les souris en plaçant l&#39;animal dans une chambre de CO 2 jusqu&#39;à ce qu&#39;il ne respirait plus. Comme seconde mesure, disloquer cervicale du cou avant de commencer la dissection. Stériliser corps entier avec de l&#39;alcool éthylique à 70%. </li><li> Retirez les deux fémurs et tibias et place des os dans du DMEM stérile. </li><li> Retirer l&#39;excès de tissu musculaire et l&#39;aide delicate lingettes jusqu&#39;à ce que les os sont parfaitement propres. </li><li> Placez les os ne intactes dans une boîte de Pétri contenant de l&#39;alcool éthylique à 70% et incuber pendant 3 minutes pour tuer les micro-organismes associés. Retirez les os et les placer dans un plat de Pétri avec du DMEM stérile. </li><li> Découpez soigneusement extrémités des os. Tenez os sur un tube conique de 50 ml vides placés sur de la glace et rincer soigneusement 10 ml de DMEM froid à l&#39;aide d&#39;une aiguille 25 G par chaque os. REMARQUE: Chaque souris donnera quatre os (2 fémurs, tibias 2). Par conséquent, on devrait donner la souris environ 40 ml de suspension cellulaire. </li><li> Centrifuger la suspension de cellules à la température ambiante pendant 10 min à 650 x g. </li><li> Au cours de cette centrifugation, préparer et stériliser par filtration des macrophages de la moelle osseuse alimentation médias qui se compose de DMEM supplémenté avec 20% L929, 10% de sérum de veau foetal, 10% de NCTC-109, 1% de Pen-Strep, 1% de HEPES, 1% de L-glutamine , 1% d&#39;acides aminés non essentiels, 0,1% de 2-mercaptoéthanol. </li><li> Resuspendre le culot résultant avec 10 ml d&#39;alimentation médias. Passez suspens cellulairesions à travers une passoire 70 um de la cellule de perturber amas de cellules et d&#39;éliminer les gros débris. </li><li> Planche 1 ml de suspension cellulaire dans 10 ml de milieu d&#39;alimentation dans des boîtes de Pétri qui sont spécifiés pour la culture de tissus (pour un total de 10 plaques). </li><li> Permettre aux cellules d&#39;adhérer à 37 ° C avec 10% de CO 2. Ajouter 5 ml de milieu d&#39;alimentation frais le jour 3 Au jour 7, remplacer complètement les médias d&#39;alimentation et les macrophages sont prêts à être utilisés. </li><li> Le jour avant l&#39;expérience, détacher les macrophages de la plaque en enlevant médias alimentation et ajouter 5 ml d&#39;une cellule douce décapage réactif à l&#39;assiette. </li><li> Incuber pendant 5-10 min à 37 ° C et éliminer les macrophages avec pipetage doux. </li><li> Cellules granulés par centrifugation à 650 g pendant 10 min. </li><li> Remettre le culot dans 1 ml de l&#39;alimentation du papier. L&#39;utilisation d&#39;une dilution de 1:20, le calcul de la concentration de macrophages par pipetage de 10 ul de suspension de cellules sur un hémocytomètre. </li><li> Utilisation de 14 mm à fond de verre des boîtes de Pétri, déposer une feuille deconcentration de 1 x 10 5 macrophages directement sur ​​le puits intérieur qui peut contenir un maximum de 200 pi, afin de prendre attention à ne pas dépasser ce volume comme le montre la figure 4. </li><li> Permettre aux cellules d&#39;adhérer à la boîte de Pétri en verre en plaçant les capsules dans un incubateur à 37 ° pendant 1 heure. </li><li> Ajouter 1-2 ml de milieu supplémenté avec alimentation LPS à 1 mg / ml et IFNg à 500 u / ml et permettent aux cellules à incuber pendant une nuit. </li></ol> 3 co-incubation de macrophages murins primaires et C. neoformans <ol><li> Le jour de l&#39;expérience, retirer 1 ml de cellules en culture pendant une nuit et culot levure inoculées par centrifugation pendant 5 min à 420 x g. </li><li> Laver les cellules 3 fois avec du PBS stérile pour éliminer les médias et les produits cryptococciques extracellulaires comme le polysaccharide glucuronoxylomannan (GXM) à la vitesse et l&#39;heure mentionnée ci-dessus. </li><li> Resuspendre le culot cellulaire dans 1 ml de PBS stérile et faire une dilution de 1: 100. Pipette 10 ul de til dilution sur un hémocytomètre et compter les cellules. Ajouter les cellules de levure à une MOI de 1: 5. Par exemple, s&#39;il existe une 10 x 5 macrophages / puits, faire une suspension de cellules de 5 x 10 6 / ml de Cn et ajouter 100 ul de cette solution à une quantité totale de 5 x 10 5 Cn. </li><li> Ajouter le volume calculé de suspension cellulaire à une cryptococcose ml de milieu d&#39;alimentation avec l&#39;anticorps mAb 18B7 à une concentration de 10 pg / ml. Incuber la suspension de cellules à la température ambiante pendant 5 min. </li><li> Retirez le support d&#39;alimentation de verre à fond plat de Pétri et laver 1x avec du PBS stérile. Ajouter 100 ul de opsonisé Cn directement à bien. </li><li> Laisser les cellules à incuber pendant 2 heures à 37 ° C avec 10% de CO 2. </li><li> Après co-incubation, vérifier au microscope que les macrophages ont intériorisé Cn. Pour être considéré comme intériorisé, les cellules cryptococciques devraient être clairement visibles dans les limites de la macrophages. </li><li> Laver boîte de Pétri 3x avec PBS stérile pour éliminer toute extracellular Cn. </li><li> Ajouter 1-2 ml d&#39;alimenter les médias sans l&#39;ajout de l&#39;anticorps monoclonal 18B7, et mettre en place microscope. </li></ol> 4 Visualisation non-lytique exocytose en temps réel utilisant Digital Light Microscopy NOTE: Pour effectuer ces essais, un microscope qui comprend une chambre qui comprend l&#39;incubation de joint livraison de CO 2 et une unité de chauffage est nécessaire. <ol><li> Avant l&#39;expérience, de pré-chauffer la chambre à incuber pendant au moins 30 min à 37 ° C. Assurez-vous que l&#39;unité de CO 2 lit 5% au début de l&#39;expérience. </li><li> Lieu fond de verre boîte de Pétri sur la plate-forme de microscope, couvrir avec du CO 2 couvercle, et fermer toutes les portes afin de s&#39;assurer qu&#39;aucune chaleur s&#39;échappe. </li><li> Utilisation de l&#39;objectif 10X avec le microscope à contraste de phase, se concentrer sur un champ libre de macrophages infectés. Sur la base de l&#39;expérience, de déterminer l&#39;objectif utilisé par le nombre de macrophages doivent être étudiés. </li><li> Configurer le logiciel de microscope à take une image toutes les 4 min pour une période de 24 heures. </li><li> Après 24 h, l&#39;expérience aura atteint la fin et un total de 361 images a été recueilli. Export de ces images comme .jpegs à une compression de 5%. Utilisation de logiciels Image J, visualiser les images d&#39;une pile visuelle à 5 images par seconde. Si le film doit être vu pour publication ou une présentation, soit l&#39;enregistrer comme un avi ou mov fichier selon le format qui fonctionne le mieux pour le spectateur. </li></ol>

<ol>
	<li>Voelz, K., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcal+Interactions+With+the+Host+Immune+System.">Cryptococcal Interactions With the Host Immune System.</a> Eukaryotic cell. 9 (6), 835-846 (2010).</li><li>Johnston, S. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcus+interactions+with+macrophages:+evasion+and+manipulation+of+the+phagosome+by+a+fungal+pathogen.">Cryptococcus interactions with macrophages: evasion and manipulation of the phagosome by a fungal pathogen.</a> Cellular microbiology. 15 (3), 403-411 (2012).</li><li>Sabiiti, W., May, R. C. <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+infection+by+the+human+fungal+pathogen+Cryptococcus+neoformans.">Mechanisms of infection by the human fungal pathogen Cryptococcus neoformans.</a> Future microbiology. 7 (11), 1297-1313 (2012).</li><li>Bliska, J. B., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+pathogenic+bacteria+and+fungi+&#8212;+a+case+of+convergent+evolution.">Intracellular pathogenic bacteria and fungi &#8212; a case of convergent evolution.</a> Nature Reviews Microbiology. 7, 165-171 (2008).</li><li>Alvarez, M., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+spread+and+massive+vacuole+formation+after+Cryptococcus+neoformans+infection+of+murine+macrophages.">Cell-to-cell spread and massive vacuole formation after Cryptococcus neoformans infection of murine macrophages.</a> BMC immunology. 8 (1), 16 (2007).</li><li>Ma, H., Croudace, J. E., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+cell-to-cell+spread+of+a+pathogenic+yeast.">Direct cell-to-cell spread of a pathogenic yeast.</a> BMC immunology. 8, 15 (2007).</li><li>Alvarez, M., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagosome+extrusion+and+host-cell+survival+after+Cryptococcus+neoformans+phagocytosis+by+macrophages.">Phagosome extrusion and host-cell survival after Cryptococcus neoformans phagocytosis by macrophages.</a> Curr Biol. 16 (21), 2161-2165 (2006).</li><li>Ma, H., Croudace, J. E., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expulsion+of+live+pathogenic+yeast+by+macrophages.">Expulsion of live pathogenic yeast by macrophages.</a> Curr Biol. 16 (21), 2156-2160 (2006).</li><li>Johnston, S. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+fungal+pathogen+Cryptococcus+neoformans+escapes+macrophages+by+a+phagosome+emptying+mechanism+that+is+inhibited+by+Arp2/3+complex-mediated+actin+polymerisation.">The human fungal pathogen Cryptococcus neoformans escapes macrophages by a phagosome emptying mechanism that is inhibited by Arp2/3 complex-mediated actin polymerisation.</a> PLoS pathogens. 6 (8), (2010).</li><li>Carnell, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+polymerization+driven+by+WASH+causes+V-ATPase+retrieval+and+vesicle+neutralization+before+exocytosis.">Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis.</a> The Journal of cell biology. 193 (5), 831-839 (2011).</li><li>Voelz, K., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+signaling+regulates+the+outcome+of+intracellular+macrophage+parasitism+by+Cryptococcus+neoformans.">Cytokine signaling regulates the outcome of intracellular macrophage parasitism by Cryptococcus neoformans.</a> Infection and immunity. 77 (8), 3450-3457 (2009).</li><li>Bain, M. J., Lewis, E. L., Okai, B., Quinn, J., Gow, A. R. N., Erwig, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-lytic+expulsion/exocytosis+of+Candida+albicans+from+macrophages.">Non-lytic expulsion/exocytosis of Candida albicans from macrophages.</a> Fungal genetics and biology. 49 (9), 677-678 (2012).</li></ol>

Les auteurs déclarent que nous n&#39;avons pas les intérêts financiers concurrents ou d&#39;autres conflits d&#39;intérêts.

Ici, nous avons décrit une méthode par laquelle les interactions fongiques macrophages peuvent être enregistrées et analysées en temps réel sur une période de 24 heures. Notre laboratoire a utilisé ce protocole pour étudier de nombreux aspects temporels de l&#39;exocytose non lytique et continuera d&#39;utiliser la microscopie en temps réel pour déterminer les composants morphologiques qui entourent ce processus. Pour cette technique pour obtenir des résultats optimaux, une atten...

Les étapes d&#39;une infection fongique entre les cellules et les macrophages cryptococcose sont bien documentés 1-3. Après que les cellules de levure sont ingérés par les macrophages, une variété d&#39;interactions peuvent se produire: le macrophage peut lyser libérer sa charge fongique dans l&#39;espace extracellulaire, ou il peut contrôler l&#39;infection par le maintien des cellules de levure dans les limites de sa membrane cellulaire 4. Cependant, il ya quelques années, un nouveau résultat a été décrit de façon indépendante par deux groupes: exocytose non lytique, un processus dans lequel un macrophage radié partie ou la totalité des cellules cryptococcose dans l&#39;environnement ou une cellule voisine et à la fois l&#39;hôte et l&#39;agent pathogène restent viables 5 -8. Plusieurs études ont tenté de comprendre les mécanismes moléculaires à l&#39;origine de cette interaction, mais nous n&#39;avons pas encore une compréhension complète sur ce qui motive ce phénomène. La capacité de capturer des images en temps réel et d&#39;analyser ensuite infecter multiplesed macrophages permet de répondre à de nombreuses questions sur les propriétés physiques entourant l&#39;exocytose non lytique en ce qui concerne le calendrier, la capacité spatiale, le changement dans la morphologie et même le stress des macrophages lors d&#39;une infection fongique. En permettant aux cellules d&#39;être logés dans un environnement qui est comparable à celle d&#39;un incubateur, on peut apercevoir la nature dynamique de l&#39;interaction de cellules macrophages-fongique. Les chercheurs ont utilisé cette technique pour étudier les différentes composantes de ce processus. Le rôle du phagosome dans ce processus a été rendu évident en utilisant une coloration fluorescente et l&#39;actine agents de blocage 9, tandis qu&#39;un autre groupe a utilisé cette méthode pour montrer que l&#39;exocytose non lytique a été bloqué dans les cellules qui ont eu le WASH nucléation des domaines protéiques supprimé 10. L&#39;effet de la signalisation de cytokine a aussi été constatée en utilisant la microscopie en temps réel 11. Ce procédé n&#39;est pas limité à C. neoformans comme il a également été observée avec Candida albicans, another pathogène fongique qui a la capacité d&#39;infecter les macrophages 12. Ces résultats sont des exemples de la façon dont cette méthode peut fournir une mine d&#39;informations pour une région que nous connaissons si peu.

<table border="0" cellpadding="0" cellspacing="0" width="976">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sabouraud Dextrose Agar</td>
			<td style="width:183px;">BD</td>
			<td style="width:227px;">DF0109-17-1</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sabouraud Dextrose Broth</td>
			<td>BD</td>
			<td>DF0382-17-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dubelcco&#39;s Modified Eagle&#39;s Medium(DMEM)</td>
			<td>Corning Cellgro</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Calf Serum ( FCS)&#160;</td>
			<td>Atlanta Biologicals</td>
			<td>S12450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NCTC 109</td>
			<td>Invitrogen</td>
			<td>21340-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES Buffer</td>
			<td>Corning Cellgro</td>
			<td>25-060-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamax</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-essential Amino Acids</td>
			<td>Corning Cellgro</td>
			<td>25-025-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985-023</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3003 Tissue Culture Petri Dishes&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>08772E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellStripper</td>
			<td>Corning Cellgro</td>
			<td>25-056-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mat-tek Glass Bottom Culture Dishes</td>
			<td>MatTek</td>
			<td>P35GC-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LPS</td>
			<td>Sigma</td>
			<td>L3137</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse IFN-g</td>
			<td>Roche</td>
			<td>NC 9222016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mAb 18B7</td>
			<td colspan="2">&#160;&#160;&#160;&#160;&#160;&#160; Non-commerical antibody produced in our lab</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axiovert 200M Microscope with Incubating Chamber</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing non-lytic exocytosis of cryptococcus neoformans from macrophages using digital light microscopy

De nombreux aspects de l&#39;infection des macrophages par Cryptococcus neoformans ont été largement étudiés et bien défini. Cependant, une interaction particulière qui n&#39;est pas clairement comprise exocytose est non lytique. Dans ce procédé, des cellules de levure sont libérés dans l&#39;espace extracellulaire par un mécanisme mal compris que les deux feuilles et le macrophage Cn viable. Ici, nous décrivons comment suivre un grand nombre de macrophages infectés individuellement pendant une période de 24 heures de l&#39;infection par microscopie de temps écoulé. Macrophages infectés sont logés dans une chambre de chauffage avec une atmosphère de CO 2 fixé à un microscope qui offre les mêmes conditions qu&#39;un incubateur de culture cellulaire. Microscopie numérique en direct peut fournir des informations sur les interactions dynamiques entre un hôte et l&#39;agent pathogène qui n&#39;est pas disponible à partir d&#39;images statiques. Être capable de visualiser chaque cellule infectée peut fournir des indices sur la façon dont les macrophages traitent les infections fongiques, et vice versa. Cette technique isa un outil puissant dans l&#39;étude de la dynamique qui se trouvent derrière un phénomène complexe.

Les images que cette technique produit peuvent être analysées dans une variété de façons de recueillir des informations entourant ce qui se passe après que les cellules ont phagocyté C. neoformans. Comme on le voit sur ​​la figure 1, il existe environ 100 qui sont soit les macrophages infectés ou non infectés. Chacun de ces macrophages donne au spectateur l&#39;occasion d&#39;étudier les interactions hôte-pathogène à un niveau très microscopique. Comme les Figures 2</s...

Watch this Scientific Journal Video about Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy at JoVE.com

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Nous décrivons comment visualiser macrophages C. neoformans (Cn) les interactions en temps réel, avec un accent particulier sur le processus d&#39;exocytose non lytique en utilisant la microscopie numérique de la lumière. En utilisant cette technique individuellement macrophages infectés peuvent être étudiées pour déterminer différents aspects du phénomène.

Visualisation non-lytique exocytose de<em&gt; Cryptococcus neoformans</em&gt; À partir de macrophages au moyen de Digital microscopie optique

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

visualizing-non-lytic-exocytosis-cryptococcus-neoformans-from

Research

JoVE Journal

Immunology and Infection

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

11.7K vues.  Albert Einstein College of Medicine.  Nous décrivons comment visualiser macrophages C. neoformans (Cn) les interactions en temps réel, avec un accent particulier sur le processus d'exocytose non lytique en utilisant la microscopie numérique de la lumière. En utilisant cette technique individuellement macrophages infectés peuvent être étudiées pour déterminer différents aspects du phénomène. 

Vidéo: Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

Visualisation cellule à cellule de transfert du VIH en utilisant des clones fluorescents du VIH et Live microscopie confocale

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Recherche

Immunologie et infection

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

Évaluation de l&#39;activité anti-fongique isolé macrophages alvéolaires par microscopie confocale

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Cholestérol des macrophages épuisement et ses effets sur la phagocytose des<em&gt; Cryptococcus neoformans</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs&#8217; mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules&#160;(SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

The goal of the present&#160;protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time&#160;analyses of the secretory granule&#39;s morphology.

Enquête mât cellules sécrétoires granulés; de biosynthèse d&#39;exocytose

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

Transformation biolistique d&#39;un fluorescent Tagged Gene dans la opportuniste pathogène fongique<em&gt; Cryptococcus neoformans</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Mesurer les réponses physiologiques de<em&gt; Drosophila</em&gt; Neurones sensoriels à Lipid Phéromones Utilisation en direct d&#39;imagerie de calcium

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Isolement, caractérisation et Purification des Macrophages dans les tissus touchés par l’Inflammation associées à l’obésité

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

Visualisation des pièges extracellulaires de Macrophage à l’aide de la microscopie confocale

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolement de Salmonella typhimurium-contenant des Phagosomes des Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Visualisation de SNARE intracellulaire traite par Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...