Los autores agradecen enormemente Pierre Bourdoncle, Thomas Guilbert y Louise Rimbault del Fondo para el Cochin Imaging. Este trabajo fue apoyado por el INSERM, CNRS y la Ligue Nationale contre le Cancer (Equipe labellisée).

1. Preparación de los linfocitos T <ol><li> Preparar ratón primaria o células T humanas como se describe 10,11. </li><li> Cultivar las células T humanas en medio completo RPMI con 10% de suero humano AB a una densidad de 2 - 3 x 10 6 células / ml. NOTA: El uso de suero de ternera fetal (FCS) en lugar de suero humano puede provocar la polarización espontánea de una gran fracción de células T humanas en cultivo. Mantener estas células en cultivo durante 4 - 5 días, y otro proceso en cualquier momento durante este período. </li><li> Mantener las células T de ratón en medio completo RPMI suplementado con 10% de FCS a una densidad celular similar. Utilice inmediatamente o al día siguiente, después de un cultivo O / N a 37 ° C en presencia de 10 ng / ml IL7. </li><li> Cultivar la línea celular T CEM humana en RPMI completo suplementado con 10% de FCS. </li></ol> 2. Estimulación de quimioquinas <ol><li> Lave 5 x 10 5 células por condición experimental en medio HBSS caliente suplementado con 10 mM Hepes. Para algunos experimentos, co-transfectar células T humanas primarias el día anterior con 2 g pmaxGFP y 8 g pcDNA3.1 + (vector vacío, control), PKCζ quinasa muerta, o plásmidos PAR6 N-terminales. </li><li> Resuspender el sedimento celular en medio cálido HBSS-Hepes (usar 0.3 ml x número de condiciones experimentales). </li><li> Distribuir las células en 1,5 ml tubos de microcentrífuga. NOTA: Por lo tanto, un tubo que corresponde a una sola condición experimental contendrá 5 x 10 5 células en 0,3 ml de medio HBSS-Hepes. Para algunos experimentos, añadir 500 nM latrunculina A o vehículo (DMSO) y se incuban las células durante 30 min antes de la estimulación de quimiocinas. </li><li> Añadir de 10 a 500 ng / ml o CCL19 de quimioquinas CXCL12 en cada tubo. NOTA: Utilizamos generalmente 10-200 quimiocina ng / ml para las células T humanas. CEM células no expresan el receptor CCR7 que une CCL19. Por lo tanto, las células CEM sólo serán capaces de responder a una estimulación CXCL12. Por otra parte, utilizar concentraciones más altas de quimioquinas (300-500 ng / ml) para polarizar las células T de ratón. </li><li> Voltear los tubos varias veces e incubar en un 37 ° C baño de agua durante 8 o 10 minutos para que las células T primarias o CEM, respectivamente. </li><li> Detener el proceso de polarización mediante la adición a cada tubo 0,3 ml de una solución caliente que contiene 2% de paraformaldehído (PFA) y Hepes 10 mM en PBS. Voltear los tubos y los incuban a 37 ° C durante 5 min. </li></ol> 3. Las tinciones <ol><li> Transferencia de las células de los tubos de microcentrífuga a citometría de flujo tubos. </li><li> Llenar completamente los tubos con una solución de RT que contiene 1% de albúmina de suero bovino (BSA), EDTA 0,5 mM y Hepes 10 mM en PBS. </li><li> Centrifugar los tubos a 460 g durante 4 min. </li><li> Desechar el sobrenadante y resuspender las células en 100 l de una solución a TA que contiene 5% de FCS en PBS. </li><li> Añadir 5 l de FITC-anti-HLA-ABC en cada tubo, vórtice, y los incuban durante 30 min a RT, lejos de la luz. </li><li> Lavar las células una vez con PBS que contiene 5% de FCS, unand una vez con PBS solo. </li><li> En RT: añadir 500 l 1% PFA, incubar durante 10 min, a continuación, lavar las células con una solución que contiene 1% de BSA, EDTA 0,5 mM y HEPES 10 mM en PBS. </li><li> Descartar el sobrenadante, llenar los tubos completamente con una solución de PBS que contenía 0,2% de BSA, 0,1% de saponina, EDTA 0,5 mM y Hepes 10 mM (tampón de permeabilización). </li><li> Lavar las células dos veces en este medio. </li><li> Voltear los tubos para eliminar el medio y Vortex a disociar el sedimento celular. </li><li> Añadir 0,25 g / ml TRITC-faloidina y / o una dilución 1: 100 de anticuerpo anti-P-ERM a cada tubo, vórtice ellos y se incuba durante 30 min a TA. </li><li> Lavar las células con el tampón de permeabilización. Incubar con un anticuerpo secundario fluorescente si es necesario. </li><li> Resuspender las células en 200 l de PBS y transferirlas a tubos de microcentrífuga. NOTA: Las células están listas para la adquisición. Alternativamente, mientras se mantiene un volumen máximo total de 200 l por tubo, añadir concentrado PFA a una final 1%concentración con el fin de preservar las tinciones si las células no están para ser usados ​​el mismo día para su posterior procesamiento. </li></ol> 4. Las imágenes Adquisición y Análisis <ol><li> Encienda el aparato (véase la Tabla de específico Reactivos / Equipo) y se deja calibrar sí de acuerdo con las instrucciones del fabricante. NOTA: El software INSPIRE se utilizó con un objetivo de 40X (0,75 NA). El software IDEAS 3,0 se utilizó para todos los cuantificaciones reportados. </li><li> Preparar una serie de ventanas de adquisición que se pueden guardar en una plantilla para futuros experimentos. Dibuja histogramas que cuantifican los valores de gradiente de RMS. De la población &quot;In Focus&quot;, seleccionado en el RMS histograma gradiente, delimitar la población &quot;célula única&quot; en un histograma de la Zona. Una vez que el tubo de microcentrífuga se ha insertado en la máquina, ajustar la potencia del láser, la puerta en las células individuales y adquirir 5000 T linfocitos. </li><li> En las Ideas blandosware, abra el archivo de adquisición y crear un histograma que mostrará el valor del valor RMS gradiente de imágenes de campo claro (Canal 1) obtenida para cada célula individual. Dibuja una puerta en el histograma gradiente RMS que considera células que presentan RMS valor de gradiente anterior 57. NOTA: El gradiente de RMS permite tener en cuenta en el análisis sólo los linfocitos T que están en el plano focal. Hemos determinado empíricamente que las células individuales que presentan un valor de gradiente RMS superior a 57 están en el plano focal, y se pueden considerar con precisión. </li><li> En el menú Análisis / Máscaras, crear una nueva máscara, llamado Erode (M01,2) de la máscara de la morfología en las imágenes de campo claro M01, erosionadas de 2 píxeles. Luego, en el Análisis / menú Características, crear una nueva característica de la zona, calculado sobre la máscara Erode (M01,2). Desde las celdas seleccionadas en la puerta anterior, abra un histograma que muestra el Área de células usando esta máscara de nueva creación. La morfología de las máscaras que están disponibles por: NOTApor defecto son mucho más grandes que el tamaño real de la célula. Por lo tanto, es más preciso para erosionar que hasta 2 píxeles. </li><li> Dibuja una puerta en las células T individuales, con exclusión de las microesferas y escombros (pequeña área) y los dobletes (grandes Area). Ajuste esta puerta en cada adquisición. NOTA: Las variaciones en el tamaño de la celda afectará a la posición de la puerta. Como las células T de ratón son más pequeños que las células T humanas que son ellos mismos más pequeño que las células CEM, el área de cada tipo de célula será proporcional a su tamaño. </li><li> Teniendo en cuenta las células individuales, en foco que fueron cerrada en los pasos anteriores, abrir un gráfico de puntos que consiste en el Raw Max Pixel en la tinción de HLA-ABC (Canal 2, eje x) como una función de la Raw Max Pixel en la faloidina tinción (Canal 4, eje y). Crear una puerta para excluir los eventos fluorescentes sin teñir o saturados. Abra dos histogramas que muestran la cruda Max Pixel para HLA-ABC (Canal 2) y phalloidin (Canal 4) coloraciones. </li><li> En el Análisis / menú Máscaras, createa nueva máscara, llamado Erode (M02,2) de la máscara de la morfología de las células HLA-ABC manchados (Canal 2), erosionadas de 2 píxeles. Luego, en el Análisis / menú Características, crear una nueva característica que consiste en el parámetro de circularidad, calculado sobre la Erode (M02,2). NOTA: Este parámetro mide la desviación de la forma de la célula de un círculo. Por lo tanto, una célula T perfectamente redonda tendrá un valor alto circularidad mientras que las células polarizadas alargadas exhibirá un bajo índice de circularidad. </li><li> Posteriormente, crear otro histograma que reportar los valores de la función de circularidad de las células previamente cerradas. Utilice este histograma para trazar directamente la distribución de un individuo, población de células de enfoque de acuerdo con el valor de la circularidad para cada celda individual. </li><li> En cuanto a la forma del histograma para las células no estimuladas, dibujar una puerta para células polarizadas, a partir del valor circularidad más bajo hasta el valor límite de la circularidad donde la mayoría de las células no estimuladasse trazan. Mire la pantalla de estadísticas para el porcentaje de células polarizadas entre la población cerrada (% Recinto). NOTA: Hemos establecido esta puerta para los valores de índice de circularidad siguientes 13. </li><li> Con el fin de mirar a la polarización de la tinción de actina en las células, trazar un histograma para el Detalle Bright valor de similitud R3, comparando la tinción de HLA-ABC (canal 2) y la tinción faloidina (Canal 4). NOTA: Cuanto más grande sea el valor de esta característica, el más superpone las dos coloraciones son. </li><li> Usando la misma estrategia de gating como antes, trazar una puerta en las células no estimuladas para la tinción segregada que muestra el porcentaje de células con tinción de actina polarizada. </li><li> Cuando se haya completado, el porcentaje de células que muestran una segregación en la distribución de la MHC de clase I y tinciones con faloidina, el porcentaje de células polarizadas junto con los valores medios de los índices de la circularidad y la similitud de todas las células ± SD se muestran en la corresppaneles estadísticas onding. </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

Usando una tecnología reciente del flujo de imágenes citometría, una estrategia rápida e informativo gating para analizar los eventos celulares y moleculares inducidos por la estimulación de quimiocinas se presenta. De un solo experimento, se pueden obtener dos tipos principales de información: los cambios en la morfología celular inducida por la estimulación de quimioquinas y la distribución subcelular de proteínas diferentes durante el proceso de polarización. Curiosamente, también se proporciona evidencia...

Las quimioquinas son pequeñas proteínas solubles que atraen a las células a ubicaciones especializados 1. Por lo tanto, participan en la correcta posición de las células en los tejidos, una función crucial en el desarrollo y la fisiología. El sistema inmunológico no es una excepción a esta regla, ya que se basa en la acción de muchos tipos de células diferentes que actúan en concierto para montar una respuesta inmune efectiva. Mediante el control de la ubicación específica de un tipo de célula inmune en un estado dado, quimiocinas son pre-requerido antes de antígenos extraños pueden ser detectados y neutralizados. En los linfocitos T, en particular, las quimioquinas se unen a receptores específicos de la superficie que, tras el acoplamiento, provocan muchas señales intracelulares (altura de calcio, la fosforilación de ERK, la activación de Rho GTPasas, el aumento de las integrinas de afinidad y alteraciones del citoesqueleto) que favorecen la motilidad celular T 2,3. A nivel celular, se puede observar alteraciones morfológicas provocados a la estimulación de quimiocina.Estos cambios en las formas de células son especialmente dramática en las células T: células T en reposo tienen una morfología redonda de cordón cuando se viaja en el torrente sanguíneo. Sin embargo, la detección de la presencia de quimiocinas en sitios de inflamación o en la proximidad de los órganos linfoides se va a cambiar la forma de las células T que ahora adoptar una morfología típica de &quot;espejo de mano&quot; que consiste en una forma bipolar: un borde de ataque en la parte delantera y un borde de salida, o urópodo, en la parte posterior 4. Además, los componentes intracelulares pueden segregar en estas dos regiones opuestas de una célula T polarizada para sostener la migración. Por ejemplo, los filamentos de actina de polimerización aumenta tras la estimulación de quimiocina 5 y actina polimerizada se acumula en la parte delantera de una célula T polarizada 2. Por otro lado, varias proteínas, tales como proteínas fosforiladas de la Ezrin-radixina-Moesin (ERM) de la familia que unen la membrana plasmática a la cortical del citoesqueleto de actina F, re-localizar en el urópodo de polarizadoLas células T 6. Curiosamente, nosotros y otros han demostrado que se requiere este proceso de polarización para la migración de células T. De hecho, cualquier tratamiento que interfiere con polarización inhibir la motilidad celular. Por ejemplo, la inhibición de la actividad de los miembros de la proteína atípica quinasas C (PKC) de la familia, la polarización celular y PKCζ PKCι bloques de T y su proceso de escaneo migratorio de las células dendríticas 7. Polarización de las células T también está regulada por Rho GTPasas. Hemos demostrado que la modulación de la actividad de RhoA por la proteína Fam65b descrito recientemente interfiere con los cambios en la morfología de células T y su capacidad para migrar en un ensayo Transwell 6. Como la polarización es un paso requisito previo para la motilidad celular, por lo que es crucial para poder cuantificar como una lectura principal de las respuestas a quimiocinas. Alteraciones de la forma de la célula se midieron previamente manualmente 8. Sin embargo, este tipo de cuantificación es muy lento, de modo que por lo general sólo unos pocosdecenas de células se toman en cuenta. Aquí, un nuevo método se presenta para cuantificar rápidamente el grado de alteraciones de la forma de los linfocitos T expuestos a quimioquinas estimulación. Un flujo de imágenes citometría de la tecnología (véase la Tabla de reactivos específicos / Equipment) se utiliza, que combina las ventajas de un citómetro de flujo y un microscopio 9 para cuantificar de manera eficiente la cantidad de células polarizadas en diferentes condiciones de estimulación de quimioquinas. Además de la cuantificación de las alteraciones morfológicas que uno puede medir la firmeza con esta tecnología, también es posible evaluar los cambios en la localización subcelular de algunas proteínas sobre la señalización de quimiocinas.

<table border="0" cellpadding="0" cellspacing="0" width="569">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Name of the material/Equipment</td>
			<td style="width:95px;">Company</td>
			<td style="width:80px;">Catalog number</td>
			<td style="width:255px;">Comments/Description (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">RPMI</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">61870-010</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Human serum AB</td>
			<td style="width:95px;">PAA</td>
			<td style="width:80px;">C11-021</td>
			<td style="width:255px;">Pre-heat to inactivate the complement.</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:140px;">Fetal calf serum</td>
			<td style="width:95px;">PAN Biotech</td>
			<td style="width:80px;">P30-3300</td>
			<td style="width:255px;">Pre-heat to inactivate the complement.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:140px;">Human T cell nucleofactor kit</td>
			<td style="width:95px;">Lonza</td>
			<td style="width:80px;">VCA-1002</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Murine IL7</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">217-17</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:140px;">HBSS</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">14025-050</td>
			<td style="width:255px;">Warm in 37 &#176;C water bath before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Hepes</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">15630-056</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:140px;">Murine CCL19</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">250-27B</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:140px;">Human CCL19</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">300-29B</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells.</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:88px;width:140px;">Human CXCL12</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">300-28A</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells. This chemokine can also be used on mouse T cells.</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:140px;">PFA</td>
			<td style="width:95px;">Electron Microscopy Sciences</td>
			<td style="width:80px;">157-8-100</td>
			<td style="width:255px;">This is a 8 % PFA solution in water. Mix volume to volume with 2X PBS to obtain a 4 % PFA solution in PBS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">&#160;BSA</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:80px;">A3059</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Saponin</td>
			<td style="width:95px;">Fluka</td>
			<td style="width:80px;">84510</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">Alexa Fluor 594 phalloidin</td>
			<td style="width:95px;">Invitrogen</td>
			<td style="width:80px;">A12381</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">FITC-anti-HLA-ABC antibody</td>
			<td style="width:95px;">Beckman Coulter</td>
			<td style="width:80px;">IM1838</td>
			<td style="width:255px;">clone B9.12.1</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">Anti-P-ERM antibody</td>
			<td style="width:95px;">Cell Signaling Technology</td>
			<td style="width:80px;">3149P</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:140px;">ImageStreamx Mark II</td>
			<td style="width:95px;">Amnis</td>
			<td style="width:80px;"></td>
			<td style="width:255px;"></td>
		</tr>
	</tbody>
</table>

rapid and robust analysis of cellular and molecular polarization induced by chemokine signaling

Las células responden a la estimulación de quimiocinas por la pérdida de su forma redonda en un proceso llamado polarización, y mediante la alteración de la localización subcelular de muchas proteínas. Las técnicas clásicas de imágenes se han utilizado para estudiar estos fenómenos. Sin embargo, se requiere la adquisición manual de de muchas células, seguido de tiempo cuantificación de la morfología y la co-localización de la tinción de decenas de células. Aquí, un método rápido y potente se describe para estudiar estos fenómenos en muestras compuestas por varios miles de células por medio de un flujo de imágenes citometría de tecnología que combina las ventajas de un microscopio con los de un citómetro. El uso de linfocitos T estimulados con CCL19 y tinción para las moléculas MHC de clase I y actina filamentosa, una estrategia gating se presenta para medir simultáneamente el grado de alteraciones de la forma y el grado de co-localización de los marcadores que se ven afectados por la señalización de CCL19. Por otra parte, esta estrategia gating nos permitió OBSERVe la segregación de actina filamentosa (en el frente) y fosforilados proteínas Ezrin-radixina-Moesin (fosfo-ERM) (en la parte trasera) en las células T polarizadas después CXCL12 estimulación. Esta técnica también es útil para observar el efecto de bloqueo en la polarización de dos elementos diferentes: la inhibición de la polimerización de la actina por un inhibidor farmacológico y expresión de mutantes de la / PKC atípicas vía de señalización Par6. De este modo, se muestra evidencia de que esta técnica es útil para analizar tanto las alteraciones morfológicas y redistribuciones de proteínas.

El primer ejemplo elegido aquí se refiere al uso de linfocitos T primarias humanas estimuladas con CCL19. Sin embargo, la misma estrategia se puede utilizar con las células T de ratón primario, líneas de células T o cualquier otro tipo de células sensibles a la estimulación de quimiocina. La estrategia gating que aquí se presenta incluye una serie de ventanas que se seleccionan los eventos enfocados, entonces las células individuales. Por último, la gama de intensidad de fluorescencia se sigue aquí como un ej...

Watch this Scientific Journal Video about Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling at JoVE.com

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

Análisis rápido y robusto de Celular y Molecular Polarización Inducida por Chemokine Señalización

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many ...

rapid-robust-analysis-cellular-molecular-polarization-induced

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JoVE Journal

JoVE Core

Cell Biology

Immunology and Infection

Unit 1

Cell Polarization and Migration

SCIENTISTS IN ACTION

8.0K vistas.  Institut Cochin. Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

Video: Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors1. This process leads to bacterial invasion of endothelial cells2. More recently, we observed an interaction of Opc with a 100kDa protein found in whole cell lysates of human cells3. We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as &alpha;-actinin. As no surface expressed &alpha;-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and &alpha;-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with &alpha;-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &amp;#945;-actinin by Confocal Imaging

Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human &alpha;-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular &alpha;-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).

La visualización y cuantificación de interacciones intracelulares de Neisseria meningitidis Humanos y α-actinina de imagen confocal

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

Investigación

Inmunología e Infección

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 ...

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

Un ensayo de bioluminiscencia de calcio para el análisis funcional de los Mosquitos ( Aedes aegypti) Y Tick ( Rhipicephalus microplus) G receptores acoplados a proteínas

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Imagen del VIH-1 sobre inducida sinapsis virológica y señalización en la bicapa lipídica sintéticas

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Una técnica de microscopía TIRF en tiempo real, imágenes simultáneas de la TCR y sus proteínas asociadas de señalización

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Análisis del daño epitelial Producido por<em&gt; Entamoeba histolytica</em&gt; Infección

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Análisis rápido de aberraciones cromosómicas en linfocitos B de ratón por PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Detección Molecular rápida y diferenciación de virus gripales A y B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

Disección señalización complejos por purificación de la afinidad de complementación Bimolecular (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is characterized by lethal complications due to plasma leakage, ascites, pleural effusion, respiratory distress, severe bleeding, and organ impairment. A few cases of DENV infection present neurological manifestations; however, studies have not explored DENV-induced neuropathogenesis further. In this study, we present a protocol to use an immunocompetent outbred ICR (Institute of Cancer Research) mouse for investigating the induction of central nervous system (CNS) infection with DENV, followed by the progression of acute viral encephalitis-like disease.

Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.