Esta investigación fue financiada en parte por los premios NIH 5T32AI07506, 5R01AI033774, 5R37AI033142, 5R01AI052733.

Todo el trabajo de los animales se realizó de conformidad con los reglamentos y directrices del Instituto de Estudios en animales en el Albert Einstein College of Medicine. 1. Crecimiento Condiciones de C. neoformans H99 (serotipo A) <ol><li> Crecer individuo C. neoformans (CN) colonias sobre placas de agar Sabouraud. </li><li> Un día antes del experimento, seleccione una colonia e inocular 10 ml de caldo de Sabouraud. </li><li> Permitir que la cultura de crecer durante la noche a 37 ° C con agitación a una velocidad de 250 rpm. </li></ol> 2 Aislamiento y preparación de Primaria hueso macrófagos de C57 / Bl6 Ratones <ol><li> La eutanasia a los ratones mediante la colocación de los animales en una cámara de CO 2 hasta que no la respiración por más tiempo. Como segunda medida, cervical dislocar el cuello antes de comenzar la disección. Esterilizar todo el cuerpo con un 70% de alcohol etílico. </li><li> Retire ambos fémures y los huesos de tibia y colocar en DMEM estéril. </li><li> Retire el exceso de tejido y músculo de usolicate limpia hasta que los huesos están completamente limpias. </li><li> Coloque los huesos intactos sólo en una placa de Petri que contiene 70% de alcohol etílico y se incuba durante 3 min para matar los microorganismos asociados. Quitar los huesos y el lugar en el plato de Petri con DMEM estéril. </li><li> Corte cuidadosamente los extremos de los huesos. Mantenga el hueso largo de un tubo cónico de 50 ml vacías colocado en hielo y enjuague cuidadosamente 10 ml de DMEM frío utilizando una aguja de 25 G a través de cada hueso. NOTA: Cada ratón producirá 4 huesos (2 fémures, tibias 2). Por lo tanto un ratón debe producir aproximadamente 40 ml de suspensión celular. </li><li> Centrifugar la suspensión celular a temperatura ambiente durante 10 min a 650 x g. </li><li> Durante esta centrifugación, preparar y esterilizar por filtración macrófagos de médula ósea alimentación de los medios de comunicación que consiste en DMEM suplementado con 20% L929, 10% de suero de ternera fetal, 10% NCTC-109, 1% de Pen-Strep, 1% HEPES, 1% de L-glutamina , 1% de aminoácidos no esenciales, 0,1% de 2-mercaptoetanol. </li><li> Resuspender el sedimento resultante con 10 ml de la alimentación de los medios de comunicación. Pase suspens celularesiones a través de un colador de 70 micras de células para alterar grupos de células y eliminar los residuos de gran tamaño. </li><li> Placa 1 ml de la suspensión celular en 10 ml de medio de alimentación en placas de Petri que se especifican para el uso de cultivo de tejidos (para un total de 10 placas). </li><li> Permitir que las células se adhieran a 37 ° C con 10% de CO 2. Añadir 5 ml de medio de alimentación fresca en el día 3 En el día 7, reemplazar por completo los medios de alimentación y los macrófagos están listos para ser utilizados. </li><li> El día antes del experimento, separar los macrófagos de la placa mediante la eliminación de los medios de alimentación y añadir 5 ml de una célula suave extracción de reactivos a la placa. </li><li> Incubar durante 5-10 minutos a 37 ° C y retirar los macrófagos con pipeteo suave. </li><li> Precipitar las células por centrifugación a 650 xg durante 10 min. </li><li> Resuspender pellet en 1 ml de la alimentación de los medios de comunicación. Uso de una dilución 1:20, calcular la concentración de los macrófagos pipeteando 10 l de suspensión de células en un hemocitómetro. </li><li> El uso de 14 mm con fondo de cristal cajas de Petri, plato unconcentración de 1 x 10 5 macrófagos directamente sobre el pozo interior que tiene una capacidad máxima de 200 l, así que tome precauciones para no exceder este volumen, como se muestra en la Figura 4. </li><li> Permitir que las células se adhieren a la placa de Petri de vidrio mediante la colocación de los platos en un 37 ° C incubadora durante 1 hr. </li><li> Añadir 1-2 ml de la alimentación de medio suplementado con LPS a 1 mg / ml y IFNg en 500 u / ml y permiten a las células se incuban durante la noche. </li></ol> 3. Co-incubación de primaria murino macrófagos y C. neoformans <ol><li> En el día del experimento, eliminar 1 ml de células de cultivo de levadura y pellet inoculados durante la noche por centrifugación durante 5 min a 420 x g. </li><li> Lavar las células 3 veces con PBS estéril para eliminar los medios de comunicación y productos criptocócicas extracelulares como el glucuronoxylomanan polisacárido (GXM) a la velocidad y el tiempo mencionado anteriormente. </li><li> Resuspender sedimento celular en 1 ml de PBS estéril y crea una dilución 1: 100. Pipeta de 10 l de tél dilución en un hemocitómetro y contar las células. Añadir células de levadura a una MOI de 1: 5. Por ejemplo, si hay 1 x 10 5 macrófagos / bien, hacer una suspensión de células de 5 x 10 6 / ml de Cn y añadir 100 ml de esta solución por un importe total de 5 x 10 5 Cn. </li><li> Añadir el volumen calculado de suspensión de células criptocócica a 1 ml de medio de alimentación con el anticuerpo mAb 18B7 a una concentración de 10 mg / ml. Incubar suspensión de células a temperatura ambiente durante 5 min. </li><li> Retire el medio de alimentación de fondo de cristal placa de Petri y se lavan con PBS 1x estéril. Añadir 100 l de opsonizado Cn directamente al bien. </li><li> Permitir que las células se incuban durante 2 horas a 37 ° C con 10% de CO 2. </li><li> Después de co-incubación, comprobar al microscopio que los macrófagos han interiorizado Cn. Para ser considerado interiorizado, células criptocócicas claramente deben ser vistos dentro de los confines de los macrófagos. </li><li> Lave 3x plato de Petri con PBS estéril para eliminar cualquier y toda extracellular Cn. </li><li> Añadir 1-2 ml de la alimentación de los medios de comunicación sin la adición de mAb 18B7, y establecer microscopio. </li></ol> 4. Visualizing no lítico exocitosis en Utilizando microscopía de luz digital en tiempo real NOTA: Para realizar estos experimentos, un microscopio que viene con una cámara de incubación adjunto que incluye CO 2 y la entrega se requiere una unidad de calentamiento. <ol><li> Antes del experimento, pre-calentar la cámara de incubación durante al menos 30 min a 37 ° C. Asegúrese de que la unidad de CO 2 lee 5% al inicio del experimento. </li><li> Lugar con fondo de cristal placa de Petri en la plataforma del microscopio, cubrir con CO 2 tapa y cierre todas las puertas para asegurar que no escapa el calor. </li><li> Usando el objetivo de 10X con el microscopio de contraste de fase en, se centran en un claro campo de macrófagos infectados. Basado en el experimento, determinar el objetivo utilizado por el número de macrófagos que ser estudiados. </li><li> Configure el software de microscopio para take una imagen cada 4 minutos durante un período de 24 horas. </li><li> Después de 24 horas, el experimento habrá llegado a su conclusión y se habrá recogido un total de 361 imágenes. Exportación de estas imágenes como .jpegs a una compresión del 5%. El uso de la imagen J software, ver las imágenes como una pila Visual a los 5 fotogramas por segundo. Si la película tiene que ser vista para su publicación o una presentación, guardarla como tampoco un avi o mov archivo dependiendo del formato funciona mejor para el espectador. </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>

Los autores declaran que no tenemos que compiten intereses financieros u otros conflictos de intereses.

Aquí hemos descrito un método por el cual las interacciones fúngicas macrófagos pueden ser registradas y analizadas en tiempo real durante un período de 24 h. Nuestro laboratorio ha utilizado este protocolo para estudiar muchos de los aspectos temporales de la exocitosis no lítico y seguirá utilizando microscopía en tiempo real para determinar los componentes morfológicos que rodean este proceso. Para esta técnica para producir resultados ideales, especial atención se debe dar a c...

Las fases de una infección por hongos entre las células criptocócica y macrófagos están bien documentados 1-3. Después de células de levadura son ingeridos por macrófagos, una variedad de interacciones pueden ocurrir: el macrófago puede lisar liberando su carga fúngica en el espacio extracelular, o podría controlar la infección por el mantenimiento de las células de levadura dentro de los confines de su membrana celular 4. Sin embargo, hace varios años un nuevo resultado fue descrita independientemente por dos grupos: exocitosis no lítico, un proceso en el que un macrófago borrado algunas o todas de las células criptocócica en el ambiente circundante o una célula vecina y ambos huésped y patógeno se mantienen viables 5 -8. Varios estudios han tratado de comprender los mecanismos moleculares detrás de esta interacción, pero todavía no tienen una comprensión completa de lo que impulsa a este fenómeno. La capacidad para capturar imágenes en tiempo real y luego analizar posteriormente múltiples Infected macrófagos permite responder a muchas preguntas sobre las propiedades físicas que rodean la exocitosis no lítico en cuanto a tiempo, la capacidad espacial, el cambio en la morfología e incluso el estrés de los macrófagos durante una infección por hongos. Al permitir que las células que se encuentran en un entorno que sea comparable a la de una incubadora, podemos vislumbrar la naturaleza dinámica de las interacciones de células de tipo macrófago por hongos. Los investigadores han utilizado esta técnica para estudiar diversos componentes de este proceso. El papel del fagosoma en este proceso se hizo evidente mediante la tinción de actina fluorescente y agentes de bloqueo 9, mientras que otro grupo utilizó este método para mostrar que la exocitosis no lítico fue bloqueada en las células que han tenido la nucleación WASH dominios de la proteína suprime 10. El efecto de la señalización de citoquinas también ha sido comprobada mediante microscopía en tiempo real 11. Este proceso no se limita a C. neoformans como también se ha observado con Candida albicans, another patógeno fúngico que tiene la capacidad de infectar a los macrófagos 12. Estos hallazgos son ejemplos de cómo este método puede proporcionar una gran cantidad de información a un área que sabemos tan poco.

<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

Muchos aspectos de la infección de macrófagos por Cryptococcus neoformans han sido ampliamente estudiados y bien definido. Sin embargo, una interacción particular que no se entiende claramente es la exocitosis no lítico. En este proceso, las células de levadura se liberan en el espacio extracelular mediante un mecanismo mal entendido que deja tanto el macrófago y Cn viable. Aquí se describe cómo seguir un gran número de macrófagos infectados individualmente para un período de infección 24 h por microscopía de tiempo transcurrido. Macrófagos infectados se encuentran en una cámara de calentamiento con una atmósfera de CO 2 unido a un microscopio que ofrece las mismas condiciones que una incubadora de cultivo celular. Microscopía digital de vivo puede proporcionar información acerca de las interacciones dinámicas entre un huésped y patógeno que no está disponible a partir de imágenes estáticas. Ser capaz de visualizar cada célula infectada puede proporcionar pistas sobre cómo los macrófagos manejan las infecciones por hongos, y viceversa. Esta técnica isa poderosa herramienta en el estudio de las dinámicas que están detrás de un fenómeno complejo.

Las imágenes que esta técnica produce pueden ser analizados en una variedad de maneras de recoger información en torno a lo que sucede después de las células han fagocitado C. neoformans. Como se ve en la Figura 1, hay aproximadamente 100 macrófagos que están infectados o no infectados. Cada uno de estos macrófagos da al espectador la oportunidad de estudiar las interacciones huésped-patógeno en un nivel muy microscópico. Como muestran las figuras 2 y 3</stro...

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

Se describe cómo visualizar macrófagos C. neoformans (Cn) interacciones en tiempo real, con especial énfasis en el proceso de exocitosis no lítico usando microscopía de luz digital. Usando esta técnica de forma individual los macrófagos infectados se puede estudiar para determinar varios aspectos de este fenómeno.

La visualización no lítico exocitosis de<em&gt; Cryptococcus neoformans</em&gt; De macrófagos Usando microscopía de luz digital

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 vistas.  Albert Einstein College of Medicine.  Se describe cómo visualizar macrófagos C. neoformans (Cn) interacciones en tiempo real, con especial énfasis en el proceso de exocitosis no lítico usando microscopía de luz digital. Usando esta técnica de forma individual los macrófagos infectados se puede estudiar para determinar varios aspectos de este fenómeno. 

Video: 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.

Visualización de la célula a célula de Transmisión del VIH utilizando clones fluorescente del VIH y la microscopía confocal en vivo

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

Investigación

Inmunología e Infección

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.

La evaluación de la actividad anti-fúngica de aislados macrófagos alveolares por microscopía confocal

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.

El colesterol de los macrófagos agotamiento y su efecto sobre la fagocitosis de<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.

Investigando mastocitos secretora gránulos; de Biosíntesis de la exocitosis

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.

Transformación biolística de un fluorescente Tagged gen en el oportunista por hongos patógenos<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.

La medición de las respuestas fisiológicas de<em&gt; Drosophila</em&gt; Las neuronas sensoriales a los lípidos feromonas Uso de Imagen de calcio en Vivo

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.

Aislamiento, caracterización y purificación de los macrófagos de los tejidos afectados por la inflamación relacionados con la obesidad

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.

Visualización de macrófagos trampas extracelular mediante Microscopía Confocal

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.

Aislamiento de Salmonella typhimurium-que contienen fagosomas de los macrófagos

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.

Visualizar el lazo intracelular del tráfico de por vida de fluorescencia microscopía de imágenes

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