This work was supported by the Deutsche Forschungsgemeinschaft by grant Z within Sonderforschungsbereich 944 &#8216;Physiology and Dynamics of Cellular Microcompartments&#8217; and HE1964/18 within priority program 1580.

1. Síntesis de 10 nm nanopartículas de oro (Gold PN) 15 <ol><li> Preparar la solución A: añadir cloruro de oro acuosa 2 ml 1% en 160 ml Milli-Q, o doblemente destilada, agua. </li><li> Preparar la solución B: añadir 8 ml 1% tri-citrato de sodio x 2 H 2 O y ácido tánico 160 l 1% en 32 ml Milli-Q, o doble destilada, agua. </li><li> Calentar la solución A y B a 60 ° C y se mezclan con agitación. Observar un color azul oscuro de inmediato. Observe el color rojo después de unos 15 minutos. A continuación, calentar hasta 95 ° C, mantener 5 minutos y enfriar la solución a temperatura ambiente. </li><li> Añadir una gota de la suspensión NP en una rejilla recubierta de carbono y dejar secar al aire. Compruebe el tamaño y la morfología de los PN mediante microscopía electrónica de transmisión. </li></ol> 2. El recubrimiento de oro PN con BSA y Etiquetado con Rodamina N-hidroxisuccinimidilo Ester (NHS) 16 <ol><li> Añadir 900 l NP de oro en un tubo Eppendorf de 1,5 ml, se centrifuga a 15.000 xg durante 30 min. Opnalmente, preparar múltiples tubos pueden a la vez. </li><li> Descartar el sobrenadante, volver a suspender el sedimento en 900 l de agua esterilizada Milli-Q y añadir 100 l 2 mg / ml de BSA / PBS, mezclar en un Vortex a 800 rpm durante 30 min. </li><li> Para eliminar el exceso de BSA, centrifugar la preparación a 15.000 xg durante 60 min. </li><li> Descartar el sobrenadante y vuelva a suspender los PN oro-BSA en 125 l de PBS, añadir 12,5 l de 1 M de bicarbonato. </li><li> Inmediatamente antes de su uso, preparar una solución de 10 mg / ml de rodamina NHS / DMSO, añadir 30 l de solución de rodamina / DMSO NHS a 1 ml de la suspensión de oro-BSA NPs. Se incuba la reacción durante 2 horas (o O / N) a TA durante 800 rpm de mezcla, evitando la exposición a la luz. </li><li> Purifica los PN-oro BSA-rodamina través de diálisis contra PBS a 4 ° C con 5 cambios de tampón durante el período de 36 - 48 h. </li><li> Para estabilizar los PN, añadir 10 l de 200 mg / BSA / PBS ml en cada uno de los PN 1 ml de oro-BSA-rodamina. Para quitar la centrífuga libre o liberado BSA-rodamina, a los 15,000 g durante 60 min. Resuspender el precipitado en 2 mg / ml de BSA / PBS, medir OD 520, y se almacena a 4 ° C, evitando la exposición a la luz. </li><li> Diluir los PN 10 veces con Milli-Q o agua doblemente destilada, añadir una gota sobre una rejilla recubierta de carbono, y dejar secar al aire. Compruebe el tamaño y la morfología de los PN mediante microscopía electrónica de transmisión (TEM). NOTA: Todos los materiales utilizados para la preparación NP fueron esterilizados y la operación se llevó a cabo en una campana de cultivo celular o en un banco junto a una llama. </li></ol> 3. Cultura de células HeLa <ol><li> Células HeLa que expresan permanentemente LAMP1-GFP se cultivan en DMEM con 10% de suero de ternera fetal (FCS) y se cultivaron a 37 ° C en una atmósfera que contiene 5% de CO 2. </li><li> Semillas de las células a una densidad de 50.000 por pocillo en un porta con cámara de 8 pocillos (ibidi) e incubar O / N. </li></ol> 4. Cultura de bacterias <ol><li> Uso Salmonella enterica serotipo Typhimurium NCTC 12023 wilde tipo d (WT) cepa. Para la comparación, utilizar cepas mutantes Δ ssaV defectuosos en el SPI2-T3SS y Δ SIFA carecen efector clave SIFA para SIF. Utilice las cepas que albergan plásmido pFPV25.1 para la expresión constitutiva de una mayor GFP. NOTA: cepas bacterianas son cultivadas de forma rutinaria en caldo Luria-Bertani (LB) con la adición de 50 mg / ml de carbenicilina (WT) y LB con la adición de 50 mg / ml de carbenicilina y / o 50 mg / ml de kanamicina (Δ ssaV, Δ SIFA ) del requerido para mantener plásmidos. </li><li> Inocular una colonia de bacterias en 3 ml de LB con los antibióticos apropiados y crecer O / N a 37 ºC bajo condiciones de agitación para la aireación, luego diluido 1:31 en LB fresco y continuar el crecimiento durante 3,5 horas. En este punto del tiempo, los cultivos alcanzan la fase logarítmica tardía y las bacterias son altamente invasivos. A &#39;tambor rodillo&#39; es conveniente para incubar cultivos en tubos de ensayo con aireación. </li></ol> 5. La infección de células HeLa porSalmonella y Gold-BSA-Rodamina PN pulso Chase-etiquetado (véase la figura 1 para el esquema) <ol><li> Mida OD 600 de las bacterias sub-cultivaron y diluir a OD 600 = 0.2 en 1 ml de PBS (~ 3 × 10 8 ufc / ml), añadir cantidades apropiadas de las bacterias a las células HeLa en portaobjetos de cámara de 8 pocillos para lograr una multiplicidad de infección (MOI) de 100. </li><li> Incubar durante 30 min en la incubadora de células, lavar 3 veces con PBS para eliminar las bacterias no internalizados (este punto de tiempo se establece como 0 horas después de la infección, o 0 hr pi). Añadir 300 l de medio de cultivo fresco que contiene 100 mg / ml de gentamicina y mantener durante 1 hora. A continuación, sustituir el medio con medio fresco que contiene 10 mg / ml de gentamicina para el resto del tiempo de incubación. </li><li> Después de la incubación con medio de cultivo que contiene 100 mg / ml de gentamicina durante 1 h, el medio se sustituye por medio de la formación de imágenes (Eagles MEM sin FCS, L-glutamina, fenol rojo y bicarbonato de sodio, con 30 mM HEPES, pH 7,4) containing 10 mg / ml de gentamicina. NPS-oro BSA-rodamina se añaden a células HeLa para obtener una concentración final de OD 520 = 0,1. NOTA: NPs Gold-BSA-rodamina También se puede añadir a las células antes de la infección, o en diversos puntos de tiempo pi </li><li> Después de 1 h de incubación, retirar el medio, lavar 3 veces con PBS, y añadir 300 l de medio de formación de imágenes fresco que contiene 10% de FCS y 10 mg / ml de gentamicina para el resto del tiempo de incubación. NOTA: La duración de la incubación puede variar dependiendo de la concentración de NP y las líneas celulares utilizadas. Para macrófagos RAW264.7, se observó que 30 min de incubación con NPs a una concentración de 520 OD = 0,05 permitido suficiente internalización. </li></ol> 6. Imaging <ol><li> Utilice un sistema de imagen confocal tal como un láser de barrido confocal (CLSM) o disco giratorio (SD) microscopio con una cámara de ambiente humidificado para imágenes de alta resolución en diferentes puntos de tiempo. </li><li> Encienda la temperatura control sistema y espere hasta que se estabilice. Optimizar los ajustes de imagen como la ampliación, la velocidad de escaneo, resolución, Z-stack etc. Utilice los ajustes de excitación / emisión apropiados para GFP y PN oro-BSA-rodamina. Para este protocolo, utilizar una velocidad de exploración de 400 Hz, resolución de 512 x 512 píxeles y tamaño de Z a paso de 0,25 micras. Excite GFP y oro-BSA-rodamina usando un láser Ar (488 nm) y un láser de HeNe (543 nm), respectivamente. Incluir un (BF) del canal de campo brillante para observar la forma de las células. Por otro sistemas de microscopía y condiciones de infección, el ajuste tiene que ajustarse en consecuencia. NOTA: Utilice el mismo láser Ar como fuente de luz de canal BF y la señal fue detectada con un detector fotomultiplicador (PMT). </li><li> En indicado puntos de tiempo pi, montar los portaobjetos de cámara de 8 pocillos que contienen células infectadas en la platina del microscopio y grabar imágenes. </li></ol> 7. Análisis de Imágenes <ol><li> Utilice microscopía software de análisis de imágenes (ver <strong&gt; Materiales y Tabla de Equipo) para analizar la remodelación del sistema de endo-lisosomal por intracelular Salmonella. Abra los datos haciendo clic en &quot;Abrir&quot; y elegir el archivo. NOTA: De forma alternativa, los paquetes de software de código abierto como ImageJ o FIJI se pueden utilizar para imagen análisis cuantitativos. </li><li> En la barra de herramientas de objetos de la vista Surpass clic en el icono <img alt="Icono" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon1.jpg" /> añadir un nuevo elemento de superficie. Hacer clic en <img alt="Icono" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> (Siguiente). </li><li> Para analizar una región de interés (ROI), seleccione un segmento de retorno de la inversión. En un área de visualización, una sección rectangular con bordes superpuestos en la imagen representa el retorno de la inversión. Introduzca los valores en las direcciones x correspondiente, y-, y campos, z, o hacer clic directamente sobre las flechas en el rectángulo de vista previa para modificar el tamaño y la posición del ROI. Luego haga clic en <img alt="Icono" fo:content-width=".1in" src<html> = &quot;/ Files / ftp_upload / 52058 / 52058icon2.jpg&quot; /&gt; (Siguiente). </li><li> Como canal de origen, seleccione el canal 3 (señal de Oro-BSA-Rodamina PN). Marque la opción suave para establecer la suavidad de la superficie resultante. Defina un valor manualmente o acepte el valor generado automáticamente. En Umbral, seleccione la opción Intensidad absoluta. </li><li> Para el ajuste de umbral, seleccione la opción manual y establecer un valor. En el área de visualización, una vista previa umbral de superficie se muestra en gris. </li><li> En la ficha Clasificar Superficies la superficie resultante se puede filtrar por diversos criterios. Un filtro por defecto es &#39;número de voxels&gt; 10&#39;, y otros filtros también se puede incluir haciendo clic en &#39;añadir&#39;. Si un filtrado no es necesario, a continuación, eliminar todos los filtros haciendo clic en el botón de eliminar. Haga clic en <img alt="Icono" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> (Siguiente). </li><li> Para completar la creación de superficie, haga clic en/files/ftp_upload/52058/52058icon3.jpg &quot;/&gt; (Finalizar). En la lista de objetos, ahora se elimina la marca de la caja para el Volumen elemento y nueva superficie creada se muestra en el área de visualización. </li><li> Exportar las estadísticas. Ahora, en la vista Superar el color de los temas varían de púrpura a rojo en función de su área. Y la mesa &#39;Números Parcela&#39; muestra la información estadística (por ejemplo, número de identificación y el área de los objetos). Exportar todas las estadísticas de superficie 1 a un archivo de Excel con un clic <img alt="Icono" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon4.jpg" /> (Guardar). </li></ol>

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No conflicts of interest declared.

El sistema de endo-lisosomal de las células de mamíferos controles importantes procesos fisiológicos, incluyendo la absorción de nutrientes, la transducción de señales mediada por la hormona, la vigilancia inmune, y la presentación de antígeno 17. Hasta ahora, una variedad de marcadores se han utilizado para el etiquetado de los estudios de la vía endocítica y seguimiento. Por ejemplo, las sondas son sondas LysoTracker acidotropic fluorescentes desarrollados por Molecular Probes (Life Technologies, ...

Nanopartículas fluorescentes (PN), incluidos los PN de metal, puntos cuánticos, polímeros PN, PN sílice, puntos de carbono, etc., han atraído una atención considerable durante las últimas décadas 1,2. En comparación con los tintes orgánicos tradicionales, NPs fluorescentes muestran propiedades químicas deseables, ópticas y mecánicas, tales como fuerte intensidad de la señal, la resistencia a photobleaching y alta biocompatibilidad 3,4. Estas ventajas que el método de elección para la detección intracelular y imágenes de células vivas hacen. Además, una variedad de NPs electrón-densos son visibles por microscopía electrónica (EM), facilitando su uso para el análisis microscópico correlacionada, que permite la combinación de células vivas de seguimiento con microscopía de luz (LM) y mayor resolución a nivel ultraestructural con EM 5. Por ejemplo, los NPs de oro han sido mucho tiempo utilizado eficientemente como biosensores en células sensibles para el diagnóstico de vida, así como en el campo de la inmuno-etiquetado 6. S recientesos estudios indican que NP de oro con diferente tamaño y forma pueden ser fácilmente captación por una gran variedad de líneas celulares y transportar de forma rutinaria a través de la vía endosomal, por lo tanto tienen un gran potencial de ser aplicado para el seguimiento de la vesícula de transporte intracelular y el sistema de etiquetado endo-7,8 lisosomal . Patógenos microbianos, como Salmonella enterica, Shigella flexneri y Listeria monocytogenes, se han desarrollado diferentes mecanismos para invadir las células huésped no fagocíticas 9. Después de ser interiorizado, los agentes patógenos, ya sean localizadas en el citosol o secuestradas en compartimentos de membrana, interactuando con su entorno de acogida y modulan estos para favorecer su propia supervivencia 10. Por ejemplo, Salmonella enterica reside y se replica dentro de un compartimiento intracelular phagosomal denomina -Con vacuola Salmonella (SCV) después de la infección 11. El SCV maduracióntráficos hacia el aparato de Golgi, sufriendo continuas interacciones con la vía endocítica, e induce la formación de estructuras tubulares extensos, tales como Salmonella filamentos inducidos (SIF), clasificación túbulos nexina, Salmonella portadoras secretora inducida por proteínas de membrana (3) SCAMP3 túbulos, etc. . 12-14. Estudiando cómo estos patógenos bacterianos manipulan las vías de la célula huésped es esencial para la comprensión de las enfermedades infecciosas. Aquí, NPs de oro-BSA-rodamina fueron utilizados como trazadores de fluidos para etiquetar el sistema de endo-lisosomal celular del huésped, y el patógeno gastrointestinal Salmonella enterica serovar Typhimurium humana (Salmonella) se utilizó como un modelo de bacteria para estudiar las interacciones del patógeno con la acoger vía endocítica. PN oro-BSA-rodamina intracelular en las células no infectadas y las células infectadas con WT Salmonella o cepas mutantes se obtuvieron imágenes de un microscopio de escaneo láser confocal (CLSM).Entonces software Imaris se utilizó para cuantificar la distribución de los NPs, lo que indica que la infección por Salmonella inducida reordenamiento extremo de los endosomas / lisosomas. Después de la descripción de este método, los experimentos análogos pueden ser diseñados para rastrear el destino a largo plazo de los NPs internalizadas y para investigar la influencia de diferentes sustancias exógenas o factores endógenos en la vía endocítica de las células eucariotas.

<table border="0" cellpadding="0" cellspacing="0" width="684">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:193px;">Name of the Material/Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:149px;">Catalog Number</td>
			<td style="width:176px;">Comments/ Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gold chloride</td>
			<td>Sigma-Aldrich</td>
			<td>520918</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tannic acid</td>
			<td>Sigma-Aldrich</td>
			<td>403040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tri-sodium citrate</td>
			<td>Sigma</td>
			<td>C8532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NHS-Rhodamine</td>
			<td>Pierce</td>
			<td>46406</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMSO&#160;</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamicin</td>
			<td>Applichem</td>
			<td>A1492</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamcyin</td>
			<td>Roth</td>
			<td>T832</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carbenicillin</td>
			<td>Roth</td>
			<td>6344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">8-well chamber slides</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td>tissue culture treated, sterile</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaris Software</td>
			<td>Bitplane</td>
			<td>version 7.6</td>
			<td>various configurations available</td>
		</tr>
	</tbody>
</table>

application of fluorescent nanoparticles to study remodeling of the endo-lysosomal system by intracellular bacteria

Nanopartículas fluorescentes (PN) con química deseable, propiedades ópticas y mecánicas son herramientas prometedoras para etiquetar orgánulos intracelulares. Aquí, presentamos un método que utiliza oro-BSA-rodamina PN para etiquetar el sistema endo-lisosomal de las células eucariotas y supervisar la manipulación de las vías celulares de acogida por el patógeno intracelular Salmonella enterica. Los parlamentos nacionales se internalizan fácilmente por las células HeLa y localizados en endosomas tardíos / lisosomas. Infección por Salmonella indujo reordenamiento de las vesículas y la acumulación de los PN en estructuras de membrana Salmonella inducidos. Desplegamos el paquete de software Imaris para análisis cuantitativos de imágenes de microscopía confocal. El número de objetos y su distribución de tamaño en las células no infectadas eran distintos de los de las células infectadas por Salmonella, lo que indica extremadamente remodelación del sistema de endo-lisosomal por WT Salmonella.

NP de oro se han generado a través de un método bien establecido a través de la reducción del ácido cloroaúrico por el citrato y ácido tánico. Como se muestra en la Figura 2A, los NPs de oro sintetizados eran casi de forma esférica con un tamaño de aproximadamente 10 nm. BSA-recubrimiento y rodamina-etiquetado no influyeron en su morfología o tamaño (Figura 2B). Se ha informado de que NPs de oro pueden ser fácilmente absorbidos por las diversas ...

Watch this Scientific Journal Video about Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria at JoVE.com

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Este artículo describe métodos para la síntesis y marcaje fluorescente de nanopartículas (NPs). Los NPs se aplicaron en los experimentos de pulso-caza para etiquetar el sistema de endo-lisosomal de las células eucariotas. La manipulación del sistema de endo-lisosomal por las actividades del patógeno intracelular Salmonella enterica fueron seguidos por imágenes de células vivas y cuantificada.

Aplicación de nanopartículas fluorescentes para estudiar Remodelación del Sistema Endo-lisosomal por bacterias intracelulares

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

application-fluorescent-nanoparticles-to-study-remodeling-endo

Research

JoVE Journal

Immunology and Infection

9.9K vistas.  Universität Osnabrück.  Este artículo describe métodos para la síntesis y marcaje fluorescente de nanopartículas (NPs). Los NPs se aplicaron en los experimentos de pulso-caza para etiquetar el sistema de endo-lisosomal de las células eucariotas. La manipulación del sistema de endo-lisosomal por las actividades del patógeno intracelular Salmonella enterica fueron seguidos por imágenes de células vivas y cuantificada. 

Video: Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Multiplex detección de bacterias en el complejo de muestras clínicas y ambientales utilizando oligonucleótidos de acoplamiento de microesferas fluorescentes

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Investigación

Inmunología e Infección

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

La purificación y la visualización de lipopolisacárido de bacterias Gram-negativas por Hot acuosa de fenol-Extracción

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Sola célula Medidas de Ruptura Vacuolar causadas por patógenos intracelulares

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Establecimiento de un<em&gt; In vitro</em&gt; Sistema para estudiar el comportamiento intracelular de<em&gt; Candida glabrata</em&gt; En humanos THP-1 macrófagos

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

La aplicación de un sistema de expresión inducible para el Estudio de la interferencia de los factores de virulencia bacteriana con Señalización Intracelular

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

Desarrollo de un sistema de entrega de ADN económico por &quot;Acufection&quot; y su aplicación a la investigación de la piel

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Sistema para la eficacia y la citotoxicidad de detección de inhibidores de focalización intracelular<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Un novedoso sistema de alimentador-libre para la producción masiva de células de asesino naturales murinos In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic purposes. However, the visualization and quantification of multiple antigens in a single tissue section using conventional chromogenic IHC is challenging. Multiplexed imaging is especially relevant in lymphoma research and diagnostics, where markers have to be interpreted in the context of a complex tumor microenvironment. Here we describe a protocol for multiplexed fluorescent IHC staining to enable the quantitative assessment of multiple targets in specific cell types of interest in lymphoma.The method covers aspects of antibody validation, antibody optimization, the multiplex optimization with markers of lymphoma subtypes, the staining of tissue microarray (TMA) slides, and the scanning of the slides, followed by data analysis, with specific reference to lymphoma. Using this method, scores for both the mean intensity of a marker of interest and the percentage positivity are generated to facilitate further quantitative analysis. Multiplexing minimizes sample utilization and provides spatial information for each marker of interest.

Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections.

La coloración de Immunohistochemical fluorescente multiplexado, por imágenes y análisis en muestras histológicas de linfoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic ...

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

Modelos de colonización vaginal murina por bacterias cultivadas anaeróbicamente

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...