1. Infection des macrophages avec H. capsulatum, C. albicans <ol><li> 16 heures avant l&#39;infection, les macrophages de semences à la densité désirée dans des plaques à 24 puits. Dans ce protocole, une densité de 3,0 x 10 5 cellules / puits a été utilisé. </li><li> Ajouter cellules fongiques en phase exponentielle à une multiplicité désirée d&#39;infection (MOI). Ce protocole peut accueillir une gamme de MOI (0,2 à 5,0). Pour l&#39;infection de macrophages RAW264.7 avec H. capsulatum, nous avons utilisé une MOI de 0,2. </li><li> Après 1,5 h pour permettre la phagocytose, les macrophages se laver trois fois avec du PBS pour éliminer les champignons extracellulaires. </li><li> Incuber pendant nombre d&#39;heures désiré avant l&#39;analyse de l&#39;échantillon. Au bas MOI (0,2 dans notre expérience), les cellules macrophages infectés restent viables pendant plusieurs jours, et des échantillons peuvent être analysés environ tous les 12-24 heures. </li></ol> 2. lyse des macrophages <ol><li> Pour libérer les champignons de cellules macrophages, retirez médias, laver 3 fois avec du PBS, etajouter 0,5 ml d&#39;eau stérile. Dans ces conditions, les macrophages et les cellules lyseront fongiques resteront intactes. </li><li> Incuber pendant 5 min à température ambiante. </li><li> Transfert lysat pour tube stérile, garder sur la glace. </li><li> Transférer 20 ul lysat dans un tube séparé, puis ajouter 20 AO ul / solution PI. Passez directement à l&#39;étape 5: &quot;Préparation des échantillons pour l&#39;analyse par cytométrie image&quot; </li></ol> 3. UFC Placage <ol><li> Effectuer dix fois dilution des lysats (de l&#39;étape 2.3) dans les médias. </li><li> Plaque 100 ul de chaque dilution, en double exemplaire, sur des plaques HMM-agarose. Incuber dans une chambre humide à 37 ° C avec 5% de CO 2 pendant 7-8 jours. </li><li> Compter manuellement colonies sur des plaques présentant un minimum de 100 et un maximum de 1.000 colonies distinctes. </li></ol> 4. Visualisation des champignons dans les macrophages vivants <ol><li> Pour collecter des macrophages vivants, laver les cellules 3 fois avec PBS. Ajouter 0,5 ml de PBS et incuber pendant 30 min à 4 ° C.</li><li> Pour supprimer les macrophages des puits de culture de tissu, une pipette doucement monter et descendre plusieurs fois. Transférer le liquide dans le tube stérile, garder sur la glace. </li><li> Transférer 20 ul d&#39;échantillon dans un tube séparé, puis ajouter 20 AO ul / solution PI. Passez directement à l&#39;étape 5: &quot;Préparation des échantillons pour l&#39;analyse par cytométrie image&quot;. </li></ol> 5. Préparation des échantillons pour l&#39;analyse par cytométrie image <ol><li> Pipeter l&#39;échantillon cible à fond, puis transférer 20 ul d&#39;échantillon dans la chambre de comptage cellulaire jetable. </li><li> Laisser les cellules sédimenter dans la chambre pendant 30 sec. </li><li> Insérez chambre de comptage dans le cytomètre en image. </li></ol> 6. Configuration de l&#39;instrument Cellometer <ol><li> Insérez les modules optiques de fluorescence: VB-535-402 et VB-660-502 dans le système et assurez-vous qu&#39;ils sont verrouillés en place. <ol><li> VB-535-402 (excitation à 475 nm, émission à 535 nm) est utilisé pour la détection et l&#39;orange d&#39;acridine GFP. </li><li> VB-660-502 (excitatisur à 540 nm, émission à 660 nm) est utilisé pour la détection de l&#39;iodure de propidium. </li></ol></li><li> Allumez le cytomètre de l&#39;image et ouvrez le logiciel qui l&#39;accompagne. </li></ol> 7. Configuration du logiciel Cellometer <ol><li> Sélectionnez le preset &quot;Type Assay» et «Type de cellules&quot; dans le Menu déroulant Assay. <ol><li> Pour viabilité, le dosage est optimisée pour l&#39;orange d&#39;acridine et de détection de l&#39;iodure de propidium. </li><li> Pour la détection des infections à Candida albicans, le dosage est optimisé pour la détection de la GFP. </li></ol></li><li> Sélectionnez «Options» sur le dessus et cliquez sur &quot;Background prendre l&#39;image&quot;, et permettre à l&#39;opération se termine. </li><li> Cliquez sur &quot;Aperçu sur fond clair image&quot;. </li></ol> 8. Acquisition d&#39;image procédure <ol><li> Insérer la chambre d&#39;échantillon préparé dans le cytomètre d&#39;image. </li><li> Utilisez le bouton de mise au point et la mise au point. </li><li> Une fois mis au point, cliquez sur &quot;Count&quot;, et permettre à l&#39;opération d&#39;acquisition d&#39;image à remplir. </li><li> Remove de la chambre de comptage jetable et l&#39;éliminer. </li></ol> 9. Analyse d&#39;images de données <ol><li> La concentration et la mesure de la viabilité <ol><li> Une fois que le comptage est terminé, la concentration et la viabilité des cellules cibles sont affichés dans la page de résultat. </li><li> Cliquez sur &quot;Exporter&quot; pour exporter les données dans FCS Express 4 pour l&#39;analyse de la population cellulaire de la GFP dans l&#39;expérience d&#39;infection à Candida albicans. </li></ol></li><li> FCS analyse express de cellules infectées de Candida albicans <ol><li> Importez le fichier &quot;. NXDAT&quot; dans FCS Express 4 et intrigue les résultats dans un histogramme de fluorescence. </li><li> Appliquer grille de la population de cellules de l&#39;histogramme pour déterminer les pourcentages de la population de cellules infectées par Candida albicans. </li></ol></li></ol>

<ol>
	<li>Worsham, P. L., Goldman, 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=Quantitative+plating+of+Histoplasma+capsulatum+without+addition+of+conditioned+medium+or+siderophores.">Quantitative plating of Histoplasma capsulatum without addition of conditioned medium or siderophores.</a> J. Med. Vet. Mycol. 26, 137-143 (1988).</li><li>Goihman-Yahr, M., Pine, L., Albornoz, M. C., Yarzabal, L., de Gomez, M. H., San Martin, B., Ocanto, A., Molina, T., Convit, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+plating+efficiency+and+estimation+of+viability+of+suspensions+of+Paracoccidioides+brasiliensis+yeast+cells.">Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells.</a> Mycopathologia. 71, 73-83 (1980).</li><li>Bhatti, M. A., Hjertstedt, J., Hahn, B. L., Sohnle, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inefficient+delivery+of+yeast+cells+as+an+explanation+for+reduced+plating+efficiency+of+Candida+albicans.">Inefficient delivery of yeast cells as an explanation for reduced plating efficiency of Candida albicans.</a> Med. Mycol. 40, 465-469 (2002).</li><li>Chang, W. L., vander Heyde, H. C., Klein, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+quantitation+of+yeast:+a+novel+technique+for+use+in+animal+model+work+and+in+vitro+immunologic+assays.">Flow cytometric quantitation of yeast: a novel technique for use in animal model work and in vitro immunologic assays.</a> Journal of Immunological Methods. 211, 51-63 (1998).</li><li>Green, L., Petersen, B., Steimel, L., Haeber, P., Current, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+determination+of+antifungal+activity+by+flow+cytometry.">Rapid determination of antifungal activity by flow cytometry.</a> Journal of Clinical Microbiology. 32, 1088-1091 (1994).</li><li>Kirk, S. M., Callister, S. M., Lim, L. C., Schell, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+susceptibility+testing+of+Candida+albicans+by+flow+cytometry.">Rapid susceptibility testing of Candida albicans by flow cytometry.</a> Journal of Clinical Microbiology. 35, 358-363 (1997).</li><li>Chan, L. L. -. Y., Lai, N., Wang, E., Smith, T., Yang, X., Lin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+detection+method+for+apoptosis+and+necrosis+measurement+using+the+Cellometer+imaging+cytometry.">A rapid detection method for apoptosis and necrosis measurement using the Cellometer imaging cytometry.</a> Apoptosis. 16, 1295-1303 (2011).</li><li>Chan, L. L. -. Y., Shen, D., Wilkinson, A. 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L., Zhong, X., Pirani, A., Lin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+kinetic+measurements+of+rare+cell+proliferation+using+Cellometer+image-based+cytometry.">A novel method for kinetic measurements of rare cell proliferation using Cellometer image-based cytometry.</a> J. Immunol. Methods. 377, 8-14 (2012).</li><li>Chan, L. L., Zhong, X., Qiu, J., Li, P. Y., Lin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellometer+Vision+as+an+alternative+to+flow+cytometry+for+cell+cycle+analysis,+mitochondrial+potential,+and+immunophenotyping.">Cellometer Vision as an alternative to flow cytometry for cell cycle analysis, mitochondrial potential, and immunophenotyping.</a> Cytom. Part A. 79, 507-517 (2011).</li><li>Hull, C. M., Johnson, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+mating+type-like+locus+in+the+asexual+pathogenic+yeast+Candida+albicans.">Identification of a mating type-like locus in the asexual pathogenic yeast Candida albicans.</a> Science. 285, 1271-1275 (1999).</li><li>Berkes, C. A., Chan, L. L., Wilkinson, A., Paradis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Quantification+of+Pathogenic+Fungi+by+Cellometer+Image-Based+Cytometry.">Rapid Quantification of Pathogenic Fungi by Cellometer Image-Based Cytometry.</a> J. Micro. Meth. 91, 468-476 (2012).</li><li>Nguyen, V. Q., Sil, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-induced+switch+to+the+pathogenic+yeast+form+of+Histoplasma+capsulatum+requires+Ryp1,+a+conserved+transcriptional+regulator.">Temperature-induced switch to the pathogenic yeast form of Histoplasma capsulatum requires Ryp1, a conserved transcriptional regulator.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (12), 4880-4885 (2008).</li><li>Wenisch, C., Linnau, K. F., Parschalk, B., Zedtwitz-Liebenstein, K., Georgopoulos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+susceptibility+testing+of+fungi+by+flow+cytometry+using+vital+staining.">Rapid susceptibility testing of fungi by flow cytometry using vital staining.</a> Journal of Clinical Microbiology. 35, 5-10 (1997).</li></ol>

Les auteurs Leo Li-Ying Chan et Benjamin Paradis sont des employés de Nexcelom Bioscience LLC.

cytométrie en image permet à l&#39;utilisateur de capturer des images de haute qualité et, à l&#39;aide de logiciels spécialisés, effectuer une quantification rapide des cellules. Un défi potentiel à l&#39;adoption de cytométrie d&#39;image dans le domaine de la pathogenèse microbienne est que les microbes soient comptabilisées sont présents dans une population mixte de cellules, dont les cellules hôtes de mammifères. Ici, nous démontrons que cytométrie d&#39;image peut être utilisé pour la quantificat...

Études de champignons pathogènes, en association avec leurs hôtes et / ou des cellules hôtes ont souvent besoin de quantification des cellules fongiques viables sur un parcours de temps ou dans des conditions différentes de l&#39;infection. Dénombrement des unités formant colonie (UFC) est la méthode standard par lequel le nombre de cellules fongiques viables a été mesurée, cependant, cette technique présente plusieurs inconvénients et limitations. Premièrement, de nombreuses espèces de champignons ont une croissance lente. La croissance des colonies visibles sur des supports solides peut prendre 1-2 semaines, ce qui ralentit considérablement le rythme de la recherche. Deuxièmement, la manipulation des échantillons durant UFC placage est un processus laborieux, depuis plusieurs dilutions doivent être étalées pour assurer un nombre dénombrable de colonies. Troisièmement, le nombre d&#39;UFC est généralement plus faible que le nombre d&#39;organismes viables plaqué parce que l&#39;efficacité de placage est bien en dessous de 100%. Par exemple, l&#39;efficacité de placage pour le champignon Histoplasma capsulatum pathogène dimorphisme peut être aussi élevée que 90%, mais sont systématiquement aussi bas que30% et sont encore plus faibles (10%) pour le fongique liée dimorphe Paracoccidioides brasiliensis 1, 2. l&#39;efficacité de placage pour Candida albicans est également soumis à la variabilité 3. Enfin, l&#39;analyse UFC représente uniquement les cellules vivantes et en divisant activement capables de créer de la croissance sur milieu solide, alors que dans de nombreuses situations, il serait utile de déterminer la présence et la concentration de cellules inactives morts et / ou métabolique. Auparavant, les méthodes de cytométrie en flux pour la quantification de plusieurs espèces de champignons pathogènes a été décrite 4-6. Toutefois, en raison de problèmes de confinement de biosécurité liés à l&#39;utilisation du niveau de biosécurité 2 (BSL2) ou pathogènes BSL3 niveau sur cytomètres partagés, l&#39;adoption de cette technique a été limitée. Comme la cytométrie en flux, la cytométrie l&#39;image est une méthode rapide et sensible de la quantification des cellules. Cependant, cytométrie d&#39;image peut être effectuée à une fraction du coût avec des résultats comparables 7 -11. Ici, nous décrivons des méthodes pour effectuer cytométrie en image de champignons pathogènes, en association avec les cellules hôtes. Nous démontrons nos méthodes utilisant deux champignons pathogènes humains: Histoplasma capsulatum et Candida albicans H.. capsulatum est un champignon pathogène dimorphisme qui provoque des maladies respiratoires, chez les humains, il pousse la levure comme herbe et se multiplie dans les macrophages alvéolaires Candida albicans est une espèce commensale de l&#39;homme qui cause parfois candidose.. Nous montrons que cytométrie d&#39;image permet une quantification rapide de ces levures, ainsi que la capacité de visualisation.

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			<td>Life Technologies</td>
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			<td>Nexcelom Bioscience</td>
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			<td>Nexcelom Bioscience</td>
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use of image cytometry for quantification of pathogenic fungi in association with host cells

Les études sur les mécanismes de la pathogenèse cellulaire de levures pathogènes telles que Candida albicans, Histoplasma capsulatum et Cryptococcus neoformans communément emploient infection des hôtes mammifères ou des cellules hôtes (c.-à macrophages), suivie par la quantification de la levure en utilisant formant colonie analyse de l&#39;unité ou la cytométrie de flux. Bien unité formant colonie énumération a été la méthode la plus couramment utilisée dans le domaine, cette technique présente des inconvénients et limitations, y compris la croissance lente de certaines espèces fongiques sur des supports solides et des rendements faibles et / ou variable placage, ce qui est particulièrement préoccupant lorsque l&#39;on compare la croissance des souches de type sauvage et mutant. La cytométrie en flux peut fournir des informations quantitatives rapide en ce qui concerne la viabilité des levures, cependant, l&#39;adoption de détection par cytométrie en flux pour les levures pathogènes a été limité à un certain nombre de raisons pratiques, y compris son coût élevé et considérations de biosécurité. Ici, nous démontrons une image-méthodologie cytométrie utilisant la Vision Cellometer (Nexcelom Bioscience, LLC) pour la quantification des levures pathogènes viables en co-culture avec des macrophages. Nos études portent sur ​​la détection de deux pathogènes fongiques humains: Histoplasma capsulatum et Candida albicans H.. capsulatum colonise les macrophages alvéolaires en reproduisant dans le phagosome des macrophages, et ici, nous évaluer quantitativement la croissance de H. levures capsulatum dans des macrophages RAW 264.7 en utilisant l&#39;acridine orange / iodure de propidium coloration en combinaison avec cytométrie d&#39;image. Notre méthode fidèlement récapitule les tendances de croissance tels que mesurés par colonie traditionnelle formant unité énumération, mais avec une sensibilité accrue de manière significative. En outre, nous évaluons directement infection des macrophages vivants avec une souche exprimant la GFP de C. albicans. Notre méthodologie offre un moyen rapide, précis et économique pour la détection et la quantification d&#39;agents pathogènes fongiques humains importants en association esprith cellules hôtes.

Nous avons utilisé le Cellometer Vision image de cytomètre pour surveiller la croissance de H. capsulatum dans les cellules macrophages. Cellules RAW 264.7 ont été infectés par H. cellules de levure capsulatum et à divers points dans le temps, des échantillons ont été soumis à AO / PI coloration suivie d&#39;une analyse par cytométrie basée sur l&#39;image. En parallèle, les échantillons ont été analysés par énumération UFC traditionnel. A chaque instant, les échantillons o...

Watch this Scientific Journal Video about Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells at JoVE.com

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Ici, nous démontrons comment cytométrie d&#39;image peut être utilisé pour la quantification des champignons pathogènes, en association avec les cellules hôtes en culture. Cette technique peut être utilisée comme une alternative à la CFU énumération.

Utilisation de la cytométrie d&#39;image pour la quantification des champignons pathogènes en association avec les cellules hôtes

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

use-image-cytometry-for-quantification-pathogenic-fungi-association

Research

JoVE Journal

Immunology and Infection

12.8K vues.  Merrimack College. Ici, nous démontrons comment cytométrie d'image peut être utilisé pour la quantification des champignons pathogènes, en association avec les cellules hôtes en culture. Cette technique peut être utilisée comme une alternative à la CFU énumération.

Vidéo: Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

L&#39;utilisation d&#39;un piège optique pour l&#39;étude des interactions hôtes pathogènes pour l&#39;imagerie cellulaire dynamique en direct

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Recherche

Immunologie et infection

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

L&#39;utilisation du modèle EpiAirway pour Caractériser long terme Interactions hôte-pathogène

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Fluorescence méthodes de microscopie pour déterminer la viabilité des bactéries en association avec des cellules de mammifères

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis comme un organisme modèle pour l&#39;évaluation de l&#39;interaction des bactéries anaérobies avec des cellules hôtes

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

L&#39;utilisation de la cytométrie de flux pour évaluer l&#39;état de la chromatine dans les cellules T

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

Multicolor Flow Cytometry-basé Quantification des mitochondries et des Lysosomes dans les cellules T

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

Un modèle avancé de murine pour la stéatohépatite non alcoolisée en association avec le diabète de type 2

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis.
This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.

Described here is an established method to determine the extent of HIV-1 restriction by the cellular inhibitory protein SAMHD1. Human myeloid lineage U937 cells are transduced with a SAMHD1 expression vector co-expressing YFP, differentiated and then challenged with HIV-RFP. The level of restriction is determined by flow cytometry analysis.

Analyse de la restriction SAMHD1 par cytométrie en flux dans les cellules myéloïdes humaines U937

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are ...

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing&#160;approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.
Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.

Système de culture automatisé destiné à être utilisé dans les essais précliniques de thérapies dirigées par l’hôte pour la tuberculose

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent ...

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

Co-culture d’organoïdes épithéliaux murins de l’intestin grêle avec des cellules lymphoïdes innées

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...