1. La infección de macrófagos con H. capsulatum, C. albicans <ol><li> 16 horas antes de la infección, los macrófagos de semillas a la densidad deseada en placas de 24 pocillos. En este protocolo, se utilizó una densidad de células de 3,0 x 10 5 / pocillo. </li><li> Añadir células de hongos en crecimiento de fase log a una multiplicidad de infección deseada (MOI). Este protocolo se puede acomodar una variedad de MOI (0,2-5,0). Para la infección de macrófagos RAW264.7 con H. capsulatum, se utilizó un MOI de 0,2. </li><li> Después de 1,5 horas para permitir la fagocitosis, los macrófagos lavar tres veces con PBS para eliminar los hongos extracelulares. </li><li> Incubar obtener el número deseado de horas antes del análisis de la muestra. En una MOI baja (0,2 en nuestro experimento), células de macrófagos infectados permanecen viables durante varios días, y las muestras pueden ser analizadas aproximadamente cada 12-24 horas. </li></ol> 2. Lisis del macrófago <ol><li> Para liberar a los hongos de los macrófagos, retire los medios, lavar 3 veces con PBS, yañadir 0,5 ml de agua estéril. En estas condiciones, los macrófagos y lisar células fúngicas permanecerán intactos. </li><li> Incubar durante 5 min a temperatura ambiente. </li><li> Traslado lisado de tubo estéril, mantener en hielo. </li><li> Transferencia de 20 l de lisado a un tubo separado, a continuación, añadir 20 l AO / solución PI. Vaya directamente al paso 5: &quot;Preparación de la muestra para el análisis de citometría de imagen&quot; </li></ol> 3. CFU Plating <ol><li> Realizar dilución de diez veces de los lisados ​​(de la etapa 2.3) en los medios de comunicación. </li><li> Placa 100 l de cada dilución por duplicado, en placas HMM-agarosa. Incubar las placas en una cámara humidificada a 37 ° C con 5% de CO 2 durante 7-8 días. </li><li> Recuento manual de colonias en las placas que muestran un mínimo de 100 y un máximo de 1.000 colonias distintas. </li></ol> 4. Visualización de hongos dentro de los macrófagos en Vivo <ol><li> Para recoger los macrófagos vivos, lavar las células 3 veces con PBS. Añadir 0,5 ml de PBS y se incuba durante 30 min a 4 ° C.</li><li> Para eliminar los macrófagos de los pocillos de cultivo de tejidos, pipetear suavemente hacia arriba y hacia abajo varias veces. Transfiera el líquido en el tubo estéril, mantener en hielo. </li><li> Transferencia de 20 l de muestra a un tubo separado, a continuación, añadir 20 l AO / solución PI. Vaya directamente al paso 5: &quot;Preparación de muestras para análisis de citometría de imagen&quot;. </li></ol> 5. Preparación de muestras para análisis de citometría de imagen <ol><li> Pipetear la muestra objetivo a fondo y después transferir 20 l de muestra en la cámara de recuento celular desechable. </li><li> Permita que las células se asienten en la cámara durante 30 segundos. </li><li> Insertar cámara de recuento en el citómetro de imagen. </li></ol> 6. Configuración del instrumento Cellometer <ol><li> Inserte la óptica de fluorescencia Módulos: VB-535-402 y VB-660-502 en el sistema y asegurarse de que están bloqueados en su lugar. <ol><li> VB-535-402 (excitación a 475 nm, emisión a 535 nm) se utiliza para el naranja de acridina y detección de GFP. </li><li> VB-660-502 (excitatien a 540 nm, emisión a 660 nm) se utiliza para la detección de yoduro de propidio. </li></ol></li><li> Encienda el citómetro de imagen y abrir el software que lo acompaña. </li></ol> 7. Configuración del software Cellometer <ol><li> Seleccione el preset &quot;Tipo de ensayo&quot; y &quot;Tipo de la célula&quot; en el menú desplegable de ensayo. <ol><li> Para la viabilidad, el ensayo se optimiza para el naranja de acridina y la detección de yoduro de propidio. </li><li> Para la detección de la infección por Candida albicans, el ensayo se optimiza para la detección de GFP. </li></ol></li><li> Seleccione &quot;Opciones&quot; en la parte superior y haga clic en &quot;Take imagen de fondo&quot;, y permitir que se complete la operación. </li><li> Haga clic en &quot;Vista previa de imagen de campo claro&quot;. </li></ol> 8. Procedimiento de adquisición de imágenes <ol><li> Inserte el módulo de la muestra preparada en el citómetro de imagen. </li><li> Utilice el botón de enfoque y ajustar el enfoque. </li><li> Una vez en el foco, haga clic en &quot;Cuenta&quot;, y permitir que la operación de adquisición de imágenes para completar. </li><li> RemoVe la cámara de recuento desechables y disponer de manera adecuada. </li></ol> 9. Análisis de los datos de imagen <ol><li> La concentración y la medición de la viabilidad <ol><li> Una vez que el recuento se ha completado, la concentración y la viabilidad de las células diana se muestran en la página de resultados. </li><li> Haga clic en &quot;Exportar&quot; para exportar los datos en FCS Express 4 para el análisis de la población de células de las buenas prácticas agrarias en el Candida albicans experimento infección. </li></ol></li><li> FCS análisis expreso de las células infectadas por Candida albicans- <ol><li> Importe el archivo &quot;. NXDAT&quot; en FCS Express 4 y la trama de los resultados en un histograma de fluorescencia. </li><li> Aplicar puerta población de células para el histograma para determinar los porcentajes de la población de células infectadas por 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. R., Patton, W., Lai, N., Chan, E., Kuksin, D., Lin, B., Qiu, J. <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+image-based+cytometry+method+for+autophagy+detection+in+living+cells.">A novel image-based cytometry method for autophagy detection in living cells.</a> Autophagy. 8, 1371-1382 (2012).</li><li>Chan, L. L., Wilkinson, A. R., Paradis, B. D., Lai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Image-based+Cytometry+for+Comparison+of+Fluorescent+Viability+Staining+Methods.">Rapid Image-based Cytometry for Comparison of Fluorescent Viability Staining Methods.</a> Journal of Fluorescence. 22, 1301-1311 (2012).</li><li>Chan, L. 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>

Los autores Leo Li-Ying Chan y Benjamin Paradis son empleados de Nexcelom Bioscience LLC.

Citometría de imagen permite al usuario capturar imágenes de alta calidad y el uso de software especializado, realizar una rápida cuantificación de células. Un reto potencial para la adopción de la citometría de imagen en el campo de la patogénesis microbiana es que los microbios a ser contadas están presentes en una población mixta de células, incluyendo las células huésped de mamífero. Aquí, demostramos que la citometría de imagen se puede utilizar para la cuantificación de levaduras patógenas viable...

Estudios de hongos patógenos en asociación con sus anfitriones y / o células huésped a menudo requieren la cuantificación de células fúngicas viables durante un transcurso de tiempo o bajo diferentes condiciones de infección. Enumeración de las unidades formadoras de colonias (UFC) es el método estándar por el cual se ha medido el número de células de hongos viables, sin embargo, esta técnica tiene varios inconvenientes y limitaciones. En primer lugar, muchas especies de hongos son de crecimiento lento. El crecimiento de colonias visibles en medios sólidos puede tardar 1-2 semanas, disminuyendo significativamente el ritmo de la investigación. En segundo lugar, la manipulación de las muestras durante chapado UFC es un proceso laborioso, ya que varias diluciones deben ser chapados para asegurar un número contable de colonias. En tercer lugar, el número de UFC es típicamente menor que el número de organismos viables sembradas debido a que la eficiencia de plaqueo es muy por debajo de 100%. Por ejemplo, la eficiencia de la galjanoplastia para el hongo dimórfico Histoplasma capsulatum patógeno puede ser tan alta como 90%, pero son habitualmente tan bajo como30% y son aún más baja (10%) para el relacionado con hongos dimorfo Paracoccidioides brasiliensis 1, 2. Eficiencia de plaqueo para Candida albicans también está sujeta a la variabilidad 3. Por último, el análisis de CFU representa sólo las células en vivo y dividiendo activamente capaces de establecer el crecimiento en medios sólidos, mientras que en muchas situaciones, sería útil para determinar la presencia y concentración de las células muertas inactivos y / o metabólicamente. Anteriormente, los métodos de citometría de flujo para la cuantificación de varias especies de hongos patógenos ha sido descrito 4-6. Sin embargo, debido a problemas de contención de bioseguridad relacionados con el uso de bioseguridad de nivel 2 (BSL2) o patógenos BSL3 nivel de citómetros de flujo compartidas, la adopción de esta técnica ha sido limitada. Al igual que la citometría de flujo, citometría de imagen es un método sensible y rápido de cuantificación celular. Sin embargo, la citometría de imagen se puede realizar a una fracción del costo con resultados comparables 7 -11. Aquí, se describen los métodos para realizar citometría de imagen de hongos patógenos en asociación con las células huésped. Demostramos nuestros métodos utilizando dos hongos patógenos humanos: Histoplasma capsulatum y Candida albicans H.. capsulatum es un hongo patógeno dimórfico que causa la enfermedad de las vías respiratorias, en los seres humanos, que crece como levadura en ciernes y se replica dentro de los macrófagos alveolares Candida albicans es una especie comensal humanos, que en ocasiones causa la candidiasis.. Se demuestra que la citometría de imagen permite una rápida cuantificación de estas levaduras, junto con la capacidad de visualización.

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

Los estudios sobre los mecanismos de la patogénesis celulares de levaduras patógenas tales como Candida albicans, Histoplasma capsulatum y Cryptococcus neoformans emplean comúnmente infección de huéspedes mamíferos o células huésped (es decir, los macrófagos), seguido por la cuantificación de levadura utilizando formadoras de colonias unidad de análisis o la citometría de flujo. Mientras unidad formadora de colonias enumeración ha sido el método más comúnmente utilizado en el campo, esta técnica tiene desventajas y limitaciones, incluyendo el crecimiento lento de algunas especies de hongos en medios sólidos y eficiencias bajas y / o variable de chapado, lo cual es de particular preocupación cuando se comparan el crecimiento de las cepas de tipo salvaje y mutante. La citometría de flujo puede proporcionar una rápida información cuantitativa con respecto a viabilidad de la levadura, sin embargo, la adopción de la detección de citometría de flujo para las levaduras patógenas se ha visto limitado por un número de razones prácticas, incluyendo su alto costo y las consideraciones de seguridad de la biotecnología. Este sentido, demuestran una imagen basada enmetodología de citometría de uso de la Visión Cellometer (Nexcelom Bioscience, LLC) para la cuantificación de levaduras patógenas viables en co-cultivo con macrófagos. Nuestros estudios se centran en la detección de dos hongos patógenos humanos: Histoplasma capsulatum y Candida albicans H.. capsulatum coloniza los macrófagos alveolares mediante la replicación dentro del fagosoma macrófagos, y aquí, nos cuantitativamente evaluar el crecimiento de H. levaduras capsulatum en RAW 264,7 macrófagos con naranja de acridina / tinción con yoduro de propidio en combinación con citometría de imagen. Nuestro método recapitula fielmente las tendencias de crecimiento, medido por unidad formadora de colonia tradicional enumeración, pero con un aumento significativo de sensibilidad. Además, se evalúa directamente la infección de los macrófagos en vivo con una cepa que expresa GFP de C. albicans. Nuestra metodología ofrece un medio rápido, preciso, y económico para la detección y cuantificación de hongos patógenos humanos importantes en asociación ingeniocélulas huésped h.

Se utilizó el Cellometer Vision imagen citómetro para controlar el crecimiento de H. capsulatum en células de macrófagos. Las células RAW 264.7 fueron infectados con H. células de levadura capsulatum y en varios puntos de tiempo, las muestras fueron sometidas a tinción AO / PI seguida de análisis de citometría basada en imágenes. En paralelo, las muestras se analizaron por enumeración tradicional UFC. En cada punto de tiempo, las muestras se incubaron en agua para lisar los macrófa...

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

Aquí, demostramos cómo citometría de imagen se puede utilizar para la cuantificación de hongos patógenos en asociación con células huésped en cultivo. Esta técnica se puede utilizar como una alternativa a la CFU enumeración.

El uso de citometría de imagen para la cuantificación de hongos patógenos en asociación con las células huésped

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 vistas.  Merrimack College. Aquí, demostramos cómo citometría de imagen se puede utilizar para la cuantificación de hongos patógenos en asociación con células huésped en cultivo. Esta técnica se puede utilizar como una alternativa a la CFU enumeración.

Video: 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.

El uso de una trampa óptica para el estudio de interacciones huésped-patógeno de imágenes dinámicas de células vivas

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

Investigación

Inmunología e Infección

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.

Utilización del modelo de EpiAirway para la caracterización de largo plazo Interacciones Huésped-Patógeno

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.

Fluorescencia métodos de microscopía para determinar la viabilidad de las bacterias en asociación con las células de mamíferos

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 como organismo modelo para la evaluación de la interacción de bacterias anaerobias con células huésped

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

El uso de la citometría de flujo para evaluar el estado de la cromatina en las células 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.

Cuantificación de basados en citometría de flujo multicolor de las mitocondrias y los lisosomas en las células 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 modelo murino avanzado para esteatohepatitis no alcohólica en asociación con diabetes tipo 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.

Análisis de la restricción SAMHD1 por citometría de flujo en células mieloides humanas 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.

Un sistema de cultivo automatizado para su uso en pruebas preclínicas de terapias dirigidas al huésped para la tuberculosis

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.

Cocultivo de organoides epiteliales del intestino delgado murino con células linfoides innatas

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