<p class="jove_content"&gt; RN fue parcialmente financiado por una Marie Curie Becas Re-integración europeas (ERG) del Séptimo Programa Marco de la Comunidad Europea, el número de proyecto 268324.</p&gt;

1. Aislamiento de PMN de sangre humana mediante centrifugación de densidad <ol><li> Dibuja 5 9 tubos ml de sangre venosa heparinizada. </li><li> Preparar de doble capa de gradientes de Ficoll (4 gradientes para 5 tubos de sangre) de la siguiente manera: Verter 12 ml de una densidad de 1,119 g / ml de solución de Ficoll en un tubo de 50 ml y cuidadosamente capa de 10 ml de una densidad de 1,077 g / ml de solución de Ficoll en la parte superior. </li><li> Diluir la sangre con un volumen igual de PBS. </li><li> Capa de la sangre diluida con cuidado en el gradiente de Ficoll de doble capa; 20-25 ml por gradiente. </li><li> Centrifugar 20 minutos a 396 xg en un rotor de cubeta oscilante, 22 ° C sin frenado. </li><li> Preparar RPMI frío que contiene 0,05% albúmina de suero humano (RPMI-HSA). También hay que preparar 9 ml estéril desionizada H 2 O. Pre-cool tanto el RPMI y H 2 O en el hielo. </li><li> Aspirar la capa de Ficoll superior que contiene el plasma (de color amarillo) y PBMC, y la segunda capa de Ficoll (color blanco) con el uso de una bomba de vacío (poner una punta de pipeta estéril enla pipeta). </li><li> Recoge los PMNs en tubos de 50 ml con una pequeña pipeta de plástico (1 tubo para las fracciones PMN de cada 2 gradientes), y colocarlos en hielo. </li><li> Añadir frío RPMI-HSA para un volumen total de 50 ml y centrifugar durante 10 min a 249 xga 4 ° C. </li><li> Retirar el sobrenadante con el uso de una bomba de vacío y agitar suavemente el sedimento (eritrocitos y PMN). </li><li> Añadir 9 ml estéril desionizada H 2 O e iniciar el temporizador. Detener el choque osmótico hiper después de 30 segundos exactamente, mediante la adición de 1 ml de PBS concentrada 10 veces. Nota: El 30 sec son muy críticos. </li><li> Añadir frío RPMI-HSA para un volumen total de 50 ml y centrifugar durante 10 min a 249 xga 4 ° C. </li><li> Retirar el sobrenadante con el uso de una bomba de vacío y se recoge el precipitado en 1 PMN tubo con un volumen definido (1-2 ml) de RPMI-HSA. </li><li> Determinar la cantidad de células y ajustar la concentración a 1,10 7 células / ml. Dependiendo del donante, el rendimiento de PMNs aislados serán los que betwees 5 x 10 6 y 3 x 10 7 PMNs de cada tubo de 9 ml de sangre </li></ol> 2. Ensayo de citometría de flujo para la evaluación de la movilización de calcio en los PMN humanos <ol><li> Las celdas de carga (5 x 10 6 células / ml) con 2 mM Fluo-3 AM en RPMI-HSA y se incuba durante 20 minutos a temperatura ambiente balanceo lentamente, utilizando por ejemplo, un agitador de plataforma oscilante, protegido de la luz. </li><li> Mientras tanto preparar una dilución en serie (por ejemplo, 3 veces) del estímulo a 10x la concentración final. PSMα3 se utiliza en este protocolo, sin embargo cualquier estímulo GPCR que actúa sobre los neutrófilos es adecuado. </li><li> Preparar inhibidor de concentrado 10x del receptor en RPMI-HSA. Cuando el inhibidor actúa sobre el estímulo (por ejemplo HDL en PSMα3) pre-incubar 25 l estímulo con un volumen igual de inhibidor durante 10 minutos a temperatura ambiente. </li><li> Lavar las células mediante la adición de 10 ml de medio RPMI-HSA y centrifugar durante 249 x g a temperatura ambiente. Resuspender las células a 5 x10 6 células / ml. </li><li> Justo antes del experimento, diluir las células a 2x10 6 células / ml en medio RPMI-HSA y añadir 200 células l por tubo de FACS. PMNs diluidas son muy frágiles. Mantenerlos demasiado tiempo a esta concentración dará lugar a la activación automática, y lo mismo ocurre con agitación vigorosa o pipeta. </li><li> Conecte el tubo a la citometría de flujo, espere 3 segundos y comenzar la adquisición. Después de un período de tiempo fijo (por ejemplo, 8 segundos), quitar el tubo y añadir rápidamente 50 l de estímulo a la muestra. Inmediatamente vuelva a colocar el tubo en el soporte de la muestra y continuar con la adquisición. </li><li> Comience con el extremo más bajo y con la mayor concentración de estímulo. Lave la aguja citómetro de flujo regular después de cada carrera con RPMI-HSA. </li><li> Analizar los datos con el software de análisis de citometría de flujo. Comparar la señal de fluorescencia promedio antes de la adición del estímulo para que después de la adición del estímulo y el uso de los controles positivos y negativos apropiados para calcular la corriente alternafuerza tivación para cada dilución medido. </li></ol> Alternativamente, cuando el inhibidor actúa sobre los receptores de pre-incubar las células con el inhibidor. 3. Análisis de microscopía de fluorescencia de las bacterias Expresión de GFP después de la fagocitosis por los PMN <ol><li> Crecer S. Aureus cepas que contienen un constructo indicador de interés durante la noche en caldo (con antibióticos cuando sea necesario para mantener el plásmido reportero). En este caso cepa MW2 contiene un PSMα constructo indicador de GFP se utiliza 10, crecido en un tubo de plástico de 50 ml con 5 ml de medio de cultivo LB. </li><li> Para eliminar todas las buenas prácticas agrarias de la expresión O / N, diluir las culturas para OD 660 0.01 y crecer a OD 660 de 0,1. Redilute la 01:30 la cultura y controlar el crecimiento hasta que el cultivo alcanza un 660 OD de 0,1. Recoge las bacterias por centrifugación y se lava una vez en DPBS. Resuspender en 1:10 del volumen original para obtener un OD 660 de 1,0 o roughly 5,10 8 UFC / ml. </li><li> Mezclar las bacterias (1.10 7 / ml) con PMN recién aislados (1.10 6 / ml) en medio RPMI-HSA (relación 10:01) en un microtubo de 1,5 ml y añadir suero humano agrupado a una concentración final de 10%. Agitar en una plataforma de agitación durante 10 min a 37 ° C para estimular la fagocitosis. Diluir los PMNs cargados de bacterias a 5.10 5 PMN / ml y pipeta de 250 l en un pozo de un 8 bien calibrada cubreobjetos. </li><li> Imagen de los PMN con bacterias en el microscopio. Un microscopio invertido equipado con un objetivo NA 40X/0.85 funciona bien y debe ser encerrado en una cámara de ambiente oscuro para mantener el medio ambiente de manera estable a 37 ° C. Adquirir imágenes para un número de posiciones predeterminadas utilizando la cámara cada 5-10 minutos, tanto en el campo brillante y el canal de GFP para seguir la producción de GFP en el tiempo. </li></ol>

<ol>
	<li>Rigby, K. M., DeLeo, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+in+innate+host+defense+against+Staphylococcus+aureus+infections.">Neutrophils in innate host defense against Staphylococcus aureus infections.</a> Semin. Immunopathol. 34, 237-259 (2012).</li><li>Voyich, J. 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=Insights+into+Mechanisms+Used+by+Staphylococcus+aureus+to+Avoid+Destruction+by+Human+Neutrophils.">Insights into Mechanisms Used by Staphylococcus aureus to Avoid Destruction by Human Neutrophils.</a> The Journal of Immunology. 175, 3907-3919 (2005).</li><li>Kobayashi, S. D., 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=Rapid+neutrophil+destruction+following+phagocytosis+of+Staphylococcus+aureus.">Rapid neutrophil destruction following phagocytosis of Staphylococcus aureus.</a> J. Innate Immun. 2, 560-575 (2010).</li><li>DeLeo, F. R., Otto, M., Kreiswirth, B. N., Chambers, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Community-associated+meticillin-resistant+Staphylococcus+aureus.">Community-associated meticillin-resistant Staphylococcus aureus.</a> The Lancet. 375, 1557 (2010).</li><li>David, M. Z., Daum, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Community-Associated+Methicillin-Resistant+Staphylococcus+aureus:+Epidemiology+and+Clinical+Consequences+of+an+Emerging+Epidemic.">Community-Associated Methicillin-Resistant Staphylococcus aureus: Epidemiology and Clinical Consequences of an Emerging Epidemic.</a> Clinical Microbiology Reviews. 23, 616-687 (2010).</li><li>Kretschmer, D., 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=Human+Formyl+Peptide+Receptor+2+Senses+Highly+Pathogenic+Staphylococcus+aureus.">Human Formyl Peptide Receptor 2 Senses Highly Pathogenic Staphylococcus aureus.</a> Cell Host &amp; Microbe. 7, 463 (2010).</li><li>Prat, C., 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=A+homolog+of+formyl+peptide+receptor-like+1+(FPRL1)+inhibitor+from+Staphylococcus+aureus+(FPRL1+inhibitory+protein)+that+inhibits+FPRL1+and+FPR.">A homolog of formyl peptide receptor-like 1 (FPRL1) inhibitor from Staphylococcus aureus (FPRL1 inhibitory protein) that inhibits FPRL1 and FPR.</a> J. Immunol. 183, 6569-6578 (2009).</li><li>Prat, C., Bestebroer, J., de Haas, C. J. C., van Strijp, J. A. G., van Kessel, K. P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Staphylococcal+Anti-Inflammatory+Protein+That+Antagonizes+the+Formyl+Peptide+Receptor-Like+1.">A New Staphylococcal Anti-Inflammatory Protein That Antagonizes the Formyl Peptide Receptor-Like 1.</a> The Journal of Immunology. 177, 8017-8026 (2006).</li><li>de Haas, C. J., 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=Chemotaxis+inhibitory+protein+of+Staphylococcus+aureus,+a+bacterial+antiinflammatory+agent.">Chemotaxis inhibitory protein of Staphylococcus aureus, a bacterial antiinflammatory agent.</a> The Journal of Experimental Medicine. 199, 687-695 (2004).</li><li>Surewaard, B. G. J., 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=Inactivation+of+Staphylococcal+Phenol+Soluble+Modulins+by+Serum+Lipoprotein+Particles.">Inactivation of Staphylococcal Phenol Soluble Modulins by Serum Lipoprotein Particles.</a> PLoS Pathog. 8, e1002606 (2012).</li><li>Kubica, 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=A+Potential+New+Pathway+for+Staphylococcus+aureus+Dissemination:+The+Silent+Survival+of+S.+aureus+Phagocytosed+by+Human+Monocyte-Derived+Macrophages.">A Potential New Pathway for Staphylococcus aureus Dissemination: The Silent Survival of S. aureus Phagocytosed by Human Monocyte-Derived Macrophages.</a> PLoS ONE. 3, e1409 (2008).</li><li>Surewaard, B. G. J., 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=Staphylococcal+alpha-phenol+soluble+modulins+contribute+to+neutrophil+lysis+after+phagocytosis.">Staphylococcal alpha-phenol soluble modulins contribute to neutrophil lysis after phagocytosis.</a> Cellular Microbiology. , (2013).</li><li>Queck, S. Y., 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=RNAIII-Independent+Target+Gene+Control+by+the+agr+Quorum-Sensing+System:+Insight+into+the+Evolution+of+Virulence+Regulation+in+Staphylococcus+aureus.">RNAIII-Independent Target Gene Control by the agr Quorum-Sensing System: Insight into the Evolution of Virulence Regulation in Staphylococcus aureus.</a> Molecular Cell. 32, 150 (2008).</li><li>Baban, C. K., 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=Bioluminescent+Bacterial+Imaging+In+Vivo.">Bioluminescent Bacterial Imaging In Vivo.</a> J. Vis. Exp. (69), e4318 (2012).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

En los métodos descritos aquí algunos pasos son muy críticos. Vamos a destacar estas aquí. Para el aislamiento de neutrófilos por centrifugación en gradiente de densidad es importante no perturbar las capas durante o después de las etapas de centrifugación. Al aspirar los neutrófilos utilizando una pipeta de plástico, asegúrese de no apretar el globo, mientras que en la capa de células, como inyectar líquido perturbará las capas. Además, inspeccione visualmente el sedimento de...

Los neutrófilos (PMNs) son fagocitos profesionales que juegan un papel clave en la respuesta inmune innata contra Staphylococcus aureus 1. La batalla constante entre el huésped y microbio ha llevado a una carrera de armamentos de ambos. Recientemente, cepas relacionadas con la comunidad (CA) de la meticilina resistente S. aureus (MRSA) se han comprobado que parece ser muy eficiente en la elusión de los neutrófilos muerte 2,3. Modulina (MEP) soluble en fenol producción excesiva de CA-MRSA se ha asociado con una mayor virulencia 4,5. Los neutrófilos humanos pueden reconocer estos MEP a través de FPR2 que conducen a la activación de esta proteína G-receptor acoplado a 6. Uno de los acontecimientos más tempranos es la movilización de las reservas intracelulares de calcio (Ca2 +). Ca 2 + actúa como un segundo mensajero para una variedad de funciones efectoras de los PMN incluyendo la desgranulación y la fagocitosis 7. Por lo tanto Ca 2 + es un indicador muy sensible de lacapacidad funcional de las MEP para activar PMNs. Para estudiar los efectos de las MEP sobre los neutrófilos, los neutrófilos frescas se aíslan y se cargan con un medio de contraste que emite fluorescencia en la movilización del calcio. En un experimento de citometría de flujo de esta rápida movilización se puede medir. El uso de este método, es posible estudiar los efectos directos de los componentes tóxicos y otro sobre los neutrófilos, y determinar la concentración más baja a la que éstos están activos. Para nosotros, es una herramienta muy útil para estudiar el efecto de muchas proteínas producidas por S. aureus implicado en la evasión inmune, como FPR2 proteína inhibidora (FLIPR) 8, FLIPR-like 7 y quimiotaxis proteína inhibidora de Staphylococcus aureus (CHIPS) 9. Todas estas proteínas se ha demostrado que inhiben la movilización de calcio en los neutrófilos por la unión al receptor que reconoce el agonista. Recientemente, nuestro grupo ha descrito que las MEP son funcionalmente inhibidos por las lipoproteínas séricas de 10 &lt;/ Sup&gt;. Estas lipoproteínas son abundantemente presente dentro de la sangre y el tejido humano, lo que indica que las MEP ejercen su función principalmente en el medio ambiente intracelular. La disponibilidad del ensayo de movilización de calcio nos permitió medir precisamente el efecto de las lipoproteínas séricas en la activación de neutrófilos por los MEP, indicado por la considerable inhibición por concentraciones muy bajas de suero. Dado que los MEP están funcionalmente inhibidos por el suero, la hipótesis de que hay una función importante para las MEP como las toxinas intracelulares. Por lo tanto, trató de determinar el papel de las MEP después de la fagocitosis. Para investigar la expresión de los PSM en el espacio intercelular, hemos construido fusiones reportero del promotor del operón psmα a GFP. Cuando estos reportero cepas de S. aureus fueron fagocitadas por los neutrófilos, la inducción de la expresión se observa mediante microscopía de fluorescencia 10. Obviamente, esta technique permite el estudio de la expresión de un gran número de genes en S. aureus u otros patógenos después de la fagocitosis. Dado que para S. aureus sobrevivir en el nicho intercelular es muy importante para superar el sistema inmune innato 11 10 12, el estudio del papel de los genes activados en este nicho es altamente relevante para la comprensión de su virulencia.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Fluo-3, AM</td>
			<td>Molecular Probes / Life Technologies</td>
			<td>F-1241</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ficoll-Paque</td>
			<td>GE Healthcare</td>
			<td>17-5442-03</td>
			<td>density 1.077 g/ml</td>
		</tr>
		<tr>
			<td>Histopaque</td>
			<td>Sigma</td>
			<td>11191</td>
			<td>density 1.119 g/ml</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco, Life Technologies</td>
			<td>52400-025</td>
			<td>contains 25 mM HEPES and L-glutamine</td>
		</tr>
		<tr>
			<td>Leica TCS SP5 microscope</td>
			<td>Leica Microsystems, The Netherlands</td>
			<td>TCS SP5</td>
			<td>objective: HCX PL APO 40X/0.85</td>
		</tr>
		<tr>
			<td>FACSCalibur</td>
			<td>BD Biosciences</td>
			<td>FACSCalibur</td>
			<td>Very important that the tube can be removed and replaced during the measurement process</td>
		</tr>
	</tbody>
</table>

studying interactions of staphylococcus aureus with neutrophils by flow cytometry and time lapse microscopy

Se presentan métodos para estudiar el efecto de modulinas solubles en fenol (PSM) y otras toxinas producidas y secretadas por Staphylococcus aureus en los neutrófilos. Para estudiar los efectos de los PSM en los neutrófilos que aislamos neutrófilos frescos utilizando centrifugación en gradiente de densidad. Estos neutrófilos se cargan con un medio de contraste que emite fluorescencia en la movilización del calcio. La activación de los neutrófilos por las MEP inicia un aumento rápido y transitorio en la concentración de calcio libre intracelular. En un experimento de citometría de flujo de esta rápida movilización se puede medir mediante el control de la fluorescencia de un colorante pre-cargado que reacciona con el aumento de la concentración de Ca2 + libre. Con este método se puede determinar la concentración PSM necesario activar los neutrófilos, y medir los efectos de los inhibidores específicos y generales de la activación de los neutrófilos. Para investigar la expresión de los PSM en el espacio intracelular, we han construido fusiones reportero del promotor del operón PSMα a GFP. Cuando estos reportero cepas de S. aureus son fagocitadas por los neutrófilos, la inducción de la expresión se puede observar mediante microscopía de fluorescencia.

Ensayo de citometría de flujo para la evaluación de la movilización de calcio en los PMN humanos La incubación de los neutrófilos con una serie de concentración de PSMα3 sintéticos resultantes en la activación rápida tal como se mide por el flujo de calcio, que se muestra por un aumento en la señal en FL-1. Pre-incubación de PSMα3 sintético con 0,01%, 0,1% o 1% de suero humano inhibió significativamente la capacidad para obtener los flujos de calcio (Figura 1). <...

Watch this Scientific Journal Video about Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy at JoVE.com

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

Se presentan métodos para estudiar el efecto de las MEP y otras toxinas secretadas por<em&gt; Staphylococcus aureus</em&gt; En los neutrófilos usando citometría de flujo y microscopía de fluorescencia.

El estudio de las interacciones de<em&gt; Staphylococcus aureus</em&gt; Con neutrófilos mediante citometría de flujo y microscopía Tiempo Lapso

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

studying-interactions-staphylococcus-aureus-with-neutrophils-flow

Research

JoVE Journal

Immunology and Infection

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

13.0K vistas.  University Medical Center Utrecht. Se presentan métodos para estudiar el efecto de las MEP y otras toxinas secretadas por Staphylococcus aureus En los neutrófilos usando citometría de flujo y microscopía de fluorescencia.

Video: Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

El uso de un flujo de goteo y la rotación del disco para reactores Staphylococcus aureus Análisis de Biofilm

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

Investigación

Inmunología e Infección

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

Subcutánea infección de Staphylococcus aureus resistentes a meticilina (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

Staphylococcus aureus Crecimiento con hemoglobina humana como una fuente de hierro

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

Un método simple y eficiente para detectar la activación del factor nuclear en neutrófilos humanos por citometría de flujo

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

La detección de nanopartículas fluorescentes Interacciones con inmunodeficiencia primaria subpoblaciones celulares por citometría de flujo

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Imaging neutrófilos y monocitos en mesentérica venas por Microscopia intravital en ratones anestesiados en Tiempo Real

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

genotipado de<em&gt; Staphylococcus aureus</em&gt; por Ribosómica espaciador de PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

Metodología para el Estudio de la transferencia horizontal de genes en<em&gt; Staphylococcus aureus</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Proyección de imagen 3D Time-lapse de la fagocitosis por macrófagos de ratón

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Un modelo de ratón para evaluar respuesta inmune innata a la infección por Staphylococcus aureus

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...