Este trabajo fue apoyado por una beca de la Fundación Nacional de Ciencia de Suiza (31003A_135278) para SSt. MOP es el beneficiario de una beca de doctorado del Fondo de Investigación AXA. Agradecemos a Patricia Nigg en busca de ayuda con el diseño de la figura 1.

Nota antes del comienzo: la campana de flujo laminar y Uso biocontención adecuada cuando se trabaja con virus vivo. A continuación, describimos las condiciones de crecimiento adecuadas a la tensión virus de la influenza cultura A / WSN / 33. Multiplicidad de infección (MOI) de incubación y tiempos pueden variar dependiendo de la cepa de virus utilizada. 1. Preparación de biotinilado A / WSN / 33 Virus <ol><li> Las células en un matraz T175 cm 2 utilizando Medio Eagle Modificado de Dulbecco (DMEM) suplementado con 10% de FBS y 1% de penicilina-estreptomicina (Pen / Strep) Madin-Darby Seed kindey canino (MDCK). Células de cultivo a 37 ° C hasta aproximadamente el 70% de confluencia se alcanza. </li><li> Antes de la infección, retire medio y lavar las células con solución salina tamponada con fosfato (PBS, pH7.0-7.3) mediante la adición de un volumen lo suficientemente grande como para cubrir la monocapa de células. </li><li> Retire PBS e infectar las células con A / WSN / 33 con una MOI de 10 -4. Diluir virus en PBS suplementado con 0,02 mg de Mg 2 +, 0,01 mg de Ca2 + &lt;/ sup&gt;, 0,3% de albúmina de suero bovino (BSA), y 1% Pen / Strep (infección-PBS). Utilice un inóculo de 4 ml para un matraz T175 cm 2. </li><li> Se incuban las células 1 hora a 37 ° C. Retire inóculo por aspiración y pipeta 10 ml de DMEM suplementado con 0,3% de BSA, 20 mM HEPES, y el 1% penicilina / estreptomicina en el matraz. </li><li> Se incuban las células a 37 ° C durante aproximadamente 3 días hasta que el efecto citopático (CPE) es visible, y después de la cosecha sobrenadante. CPE puede ser reconocido como el redondeo de las células seguido de desprendimiento y muerte de las células 14. </li><li> Girar sobrenadante a 450 xg durante 5 min para eliminar restos celulares. Transferir el sobrenadante a un nuevo tubo Falcon y repita este paso una vez más. Deseche los pellets. </li><li> Sobrenadantes de transferencia en tubos de ultracentrífuga y sobrenadantes de capas de base con 5 ml de sacarosa al 30% diluido en tampón NTE (10 mM de Tris [pH 7,4], NaCl 100 mM, EDTA 1 mM). Equilibre los tubos antes de la ultracentrifugación y se llenan con PBS para alcanzar un volumen final de 30 ml. Girar supernatantes en 112.398 xg (correspondiente a 25.000 rpm para un rotor SW28) durante 90 min en una ultracentrífuga. </li><li> Retire con cuidado el sobrenadante con la pipeta y disfrutar sedimento de virus durante la noche a 4 ° C en 250 l de PBS. Resuspender pellet pipeteando arriba y abajo. Mida el contenido de proteínas del virus purificado por Bradford ensayo 15. </li><li> Utilice un kit de biotinilación para generar biotina A / WSN / 33. Siga las instrucciones del fabricante. </li><li> En resumen, añadir la cantidad apropiada de biotina para el virus purificado y se incuba durante 2 h en hielo. Transferencia de virus a un tubo de ultracentrífuga y llenar el tubo con PBS para alcanzar un volumen final de 30 ml. Haga girar virus a 112.389 xg durante 90 minutos en una ultracentrífuga. Aspirar el sobrenadante con la pipeta y añadir 250 l de PBS. Remoje pellet durante la noche a 4 ° C. Alícuota y almacenar el virus biotinilado se almacenó a -80 ° C donde va a ser estable durante al menos 24 meses. NOTA: Puede ser útil para determinar el título de la p biotiniladoreparación de IAV por ensayo de placa estándar. Sin embargo, debe tenerse en cuenta que el protocolo posterior sólo medir partículas de virus biotinilado. Restante partículas no biotinilados debido a biotinilación incompleta contribuirá a la título infeccioso mediante ensayo en placa pero no a la señal medida en el ensayo de unión / internalización. </li></ol> 2. Determinar la cantidad necesaria de Biotinylated Virus NOTA: Antes de realizar el ensayo de unión / internalización, es importante para determinar la cantidad de virus requerida para infectar biotinilado todas las células de una muestra. <ol><li> Cultura y células A549 trypsinize de acuerdo con las instrucciones del fabricante. Contar las células por medio de una cámara de celular. Pipeta células 200.000 A549 en FACS Deepwell tubos. Spin 3 min a 450 x g. Eliminar el sobrenadante y resuspender el pellet con 200 l de PBS. Hacia abajo en hielo durante 10 min Super células. </li><li> Preparar 5 veces diluciones seriadas del biotistock de virus en la infección por nylated-PBS. </li><li> Centrifugar los tubos que contienen las células a 4 ° C durante 3 min a 450 x g. Eliminar el sobrenadante y resuspender el pellet con 100 l infección PBS que contiene el virus. Como el uso de control de la infección maqueta-PBS sin virus biotinilado. Incubar los tubos en hielo durante 1 hr. </li><li> Centrifugar los tubos a 4 ° C durante 3 min a 450 x g. Eliminar el sobrenadante y resuspender el pellet en 200 l de PBS. Repita este paso una vez más. </li><li> Para la fijación, tubos de centrifugado a 4 ° C durante 3 min a 450 x g. Eliminar sobrenadante y el sedimento se resuspende en 100 l de 3,7% de paraformaldehído (PFA). Incubar durante 10 min a TA. A continuación, repita el paso de lavado descrito en 2.4). </li><li> Para permeabilización, tubos de centrifugado a 4 ° C durante 3 min a 450 x g. Eliminar el sobrenadante y resuspender el pellet en 100 l de PBS que contiene 0,5% de Triton X-100. Incubar durante 6 min a TA. A continuación, repita el paso de lavado descrito en 2.4). </li><li> Centrifugar los tubos a 4 ° C durante 3 min a 450 x g. Eliminar el sobrenadante y resuspender pellet en 50 l 2% de BSA diluido en PBS que contenía STV-Cy3. (Tenga en cuenta que la concentración apropiada de STV-Cy3 debe determinarse antes de iniciar el experimento Comience con una concentración de 1:. 200 y la prueba 2 veces diluciones de hasta 1:. 2400 En nuestras manos, una dilución de 1: 1000 probado a ser óptimo.) </li><li> Incubar 1 hora en la oscuridad a temperatura ambiente. A continuación, repita el paso de lavado descrito en 2.4, pero resuspender el precipitado en 200 l 2% de BSA diluido en PBS. </li><li> Analizar muestras por citometría de flujo 16. Puerta de la población Cy3-positivo. Elige la concentración más baja de virus biotinilado resultante en el número máximo de células Cy3-positivas en la población A549. </li></ol> 3. Determine la cantidad necesaria de estreptavidina NOTA: La eficiencia de bloquear la señal derivada de virus extracelular determina el rango de detección del ensayo. Por lo tanto, es importante que se suprime la señal de STV-Cy3 tanto como sea posible. The cantidad necesaria de STV necesario depende de la cantidad de stock de virus biotinilado utilizado (determinado en la sección 2). <ol><li> Siga los pasos 2.1-2.4, pero el uso de concentración del virus determina en el apartado 2. </li><li> Preparar diluciones seriadas de cinco veces de STV diluidos en PBS que contenía 2% de BSA y 0,1% de azida de sodio. </li><li> Centrifugar los tubos a 4 ° C durante 3 min a 450 x g. Eliminar sobrenadante y el sedimento se resuspende en 50 l STV dilución. Como el uso de control de simulacro de PBS que contenía BSA al 2% y el 0,1% de azida de sodio y sin STV. Incubar los tubos durante 30 min en hielo. A continuación, repita paso de lavado descrito en 2.4. </li><li> Siga los pasos 2,5-2,9. Para el ensayo de internalización, elegir la concentración más baja de STV que resulta en un fuerte bloque de la señal de Cy3 (en comparación con la muestra en la que no estaba presente STV). Para ello, el porcentaje de células Cy3-positivo o la intensidad media de fluorescencia de la señal de Cy3 se puede utilizar. Idealmente, la señal de Cy3 de muestras tratadas con STV debe ser lo más cercano a la señal de infectados por el simulacrocélulas como sea posible. </li></ol> 4. Adjunto / Ensayo Internalización NOTA: Asegúrese de preparar todos los reactivos antes de comenzar el ensayo. Para los tres grupos de control que se presentan en la sección de resultados (células infectadas simuladas, las células pre-tratadas de la neuraminidasa y células azida sódica tratados) pequeñas alteraciones en el protocolo son obligatorios. <ol><li> Preparar 16 tubos para pozos profundos que contienen cada 200.000 células A549. Al lado del conjunto experimental que consta de 4 tubos, hay 3 condiciones de control, cada uno que requieren 4 tubos: las células infectadas, la neuraminidasa (NA) células -pretreated y células tratadas con azida de sodio. Cada grupo consta de 4 tubos etiquetados como &quot;0 min&quot;, &quot;0 min + STV &#39;, &#39;30 minutos&#39;, &#39;30 minutos + STV &#39;. </li><li> Centrifugar los tubos a 4 ° C durante 3 min a 450 x g. Eliminar el sobrenadante y resuspender el pellet en 200 l de PBS. </li><li> Centrifugar los tubos a 4 ° C a 450 x g. Eliminar el sobrenadante y resuspender pellet 200 en DMEM que contiene 0,3 l% de BSA, HEPES 20 mM y 1% (medio de incubación) penicilina-estreptomicina. Incubar durante 30 minutos a 37 ° C. NOTA: Para el control de la neuraminidasa: Uso neuraminidasa bacteriana (por ejemplo, la sialidasa de Vibrio cholera) a una concentración de 200 mU / ml diluido en medio de incubación para resuspender las células. </li><li> Centrifugar los tubos a 4 ° C durante 3 min a 450 x g. Eliminar el sobrenadante y resuspender el pellet en 200 l de PBS. Repita este paso una vez más, a continuación, los tubos se enfríe en hielo durante 10 min. </li><li> Centrifugar los tubos a 4 ° C durante 3 min a 450 x g. Eliminar sobrenadante y el sedimento se resuspende en 100 l virus biotinilado (utilizar la concentración de virus determina en el apartado 2 diluido en la infección-PBS). Incubar 1 hora en hielo. A continuación, repita paso de lavado descrito en 2.4. NOTA: Para el control de modelo de infectados: Uso infección PBS sin virus. Para el control de azida de sodio: Uso virus diluido en PBS-infección suplementado con 0,1% de azida de sodio. </li><li> Proprocesamiento de las muestras después de la infección en hielo: <ol><li> Para las muestras &#39;0 min&#39;, siga el paso 2.5 y almacenar las muestras a 4 ° C hasta que se fijan las otras muestras. </li><li> Para el &#39;0 min + STV&#39; muestras, tubos de centrifugado a 4 ° C durante 3 min a 450 x g. Eliminar sobrenadante y el sedimento se resuspende en 50 l STV (utilizar la concentración determinada en la sección 3) diluido en PBS que contenía BSA al 2% y el 0,1% de azida de sodio (para evitar la internalización durante la manipulación de la muestra). Incubar 30 min en hielo. Siga paso 2,4-2,5 y almacenar las muestras a 4 ° C. </li><li> Para las muestras de &#39;30 minutos &#39;, tubos de centrifugado a 4 ° C durante 3 min a 450 x g. Eliminar el sobrenadante y resuspender el pellet en 200 l de PBS que contienen 2% de BSA. Incubar 30 minutos a 37 ° C. Siga paso 2,4-2,5 y almacenar las muestras a 4 ° C. NOTA: Para el control de azida de sodio: Uso PBS suplementado con 2% de BSA y 0,1% de azida de sodio para la incubación de 30 min a 37 ° C. </li><li> Para el &#39;30 min + muestras STV &#39;giran tubos a 4 ° C durante 3 min a 450 x g. Eliminar el sobrenadante y resuspender el pellet en 200 l de PBS que contienen 2% de BSA. Incubar 30 minutos a 37 ° C. Entonces, los tubos de centrifugado a 4 ° C durante 3 min a 450 x g. Eliminar sobrenadante y el sedimento se resuspende en 50 l STV (utilizar la concentración determinada en la sección 3) diluido en PBS que contenía BSA al 2% y el 0,1% de azida de sodio (para evitar la internalización durante la manipulación de la muestra). Incubar 30 min en hielo. Siga paso 2,4-2,5 y almacenar las muestras a 4 ° C. NOTA: Para el control de azida de sodio: Uso PBS suplementado con 2% de BSA y 0,1% de azida de sodio para la incubación de 30 min a 37 ° C. </li></ol></li><li> Siga los pasos 2.6-2.8 y analizar muestras por citometría de flujo. Determinar el porcentaje de células Cy3-positivos en cada muestra. </li><li> Calcular el porcentaje adjunta virus y la cantidad relativa de virus internalizado. <ol><li> Para calcular la cantidad de virus adjunto, utilice el porcentaje de Cy3-positive células cerradas o la intensidad de fluorescencia media de la señal de Cy3. Para la comparación, establezca las células no tratadas a 100% (Figura 4B). NOTA: El porcentaje de virus adjunto se describe por la muestra &quot;0 min&quot;. </li><li> Para calcular la cantidad de virus interiorizado, tomar la relación entre el &quot;30 min + STV&quot; muestra al &quot;30 minutos&quot; de la muestra (Figura 5B). NOTA: Como se mencionó anteriormente, el porcentaje de células cerradas positivos Cy3 o la intensidad media de fluorescencia de la señal de Cy3 se puede utilizar. </li></ol></li></ol>

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The authors have nothing to disclose.

Nuestro protocolo describe una manera fácil de medir la fijación del virus y la internalización por citometría de flujo. Se permite el uso de virus de tipo salvaje marcado que imita más estrechamente las infecciones de virus en comparación con el uso de partículas similares a virus (VLP). Mientras que nuestro protocolo se ha optimizado para medir unión e internalización de IAV puede ser fácilmente adaptado para otros virus. Además, como la citometría de flujo se utiliza para la lectura, los compañeros de ti...

La entrada de los virus de influenza A (IAV) es un proceso de múltiples pasos que comienza con la unión del virus a los receptores en la membrana plasmática de las células diana 1. El receptor para IAV es el ácido siálico que está presente en una gran variedad de glicoproteínas y glicolípidos. La hemaglutinina (HA) de proteína de IAV que está presente en la envoltura viral se une al ácido siálico y por lo tanto media en la unión de las partículas virales a la membrana plasmática de las células diana 2. El virus entra en las células a través de endocitosis mediada por clatrina, sino también a las vías de entrada alternativos, como macropinocitosis, se han descrito 6.3. La interacción entre HA y ácido siálico parece ser suficiente para mediar tanto, el apego y desencadenar la internalización de las partículas virales 7. Sin embargo, se han propuesto los receptores de ingreso alternativas y sus roles en la entrada IAV están siendo investigados 1,8,9. Canallatualmente, se hacen esfuerzos para identificar los factores del huésped que están involucrados en el ciclo de vida viral con el objetivo de desarrollar urgentemente necesarias nuevas terapias antivirales 10-12. La entrada del virus sería un paso favorable para orientar el crecimiento IAV con el fin de bloquear la infección viral en su punto más temprano. Medición de las diferentes etapas de la entrada IAV experimentalmente es un reto ya que se requieren por lo general grandes cantidades de virus para detectar virus entrante. Además, el rango de detección de los cambios en la entrada del virus es solamente lineal debido a la falta de replicación viral. Esto pone de relieve la necesidad de ensayos con alta sensibilidad. El ensayo que aquí presentamos permite la detección de virus unido a la superficie celular, así como la detección de virus internalizado en relación con la cantidad total de virus asociado a células. El uso de virus de tipo salvaje biotina permite la medición conveniente a través de tinción con STV-Cy3 y lectura por citometría de flujo. Virus biotinilado es frío ligado a la células para permitir la unión, sino evitar la internalización de las partículas virales. Las células pueden ser fijos, permeabilizaron y se tiñeron con STV-Cy3 para medir virus adjunto. La señal procedente de viriones extracelulares, adjuntos puede ser derogada si se aplica una etapa de bloqueo antes de la fijación en la que las células se incuban con estreptavidina no marcada (STV). En un siguiente paso, después de la fijación de biotina IAV, la temperatura se desplaza a 37 ° C y la internalización de las partículas virales se dejó que tuviera lugar. Partículas internalizadas están protegidos de la unión de STV permitiendo así que la discriminación entre virus extra e intracelulares. Por condición experimental, se requieren cuatro muestras: &#39;0 min&#39;: La primera muestra, con la etiqueta &quot;0 min &#39;, es medir el virus frío con destino en la superficie celular. &#39;0 min + STV&#39;: La segunda muestra, con la etiqueta &quot;0 min + STV &#39;, da la intensidad de la señal de referencia del experimento. Virus adjunto se bloquea ingenioh STV y la señal después de la tinción con STV-Cy3 deberían ser mucho menor en comparación con la muestra &#39;0 min&#39;. &#39;30 Minutos &#39;: La tercera muestra, &#39;30 minutos&#39; contiene adjunta y el virus internalizado debido al cambio de temperatura a 37 ° C durante 30 minutos. &#39;30 Min + STV &#39;: La cuarta muestra, &#39;30 minutos + STV&#39; mide la fracción intracelular de virus. La etapa de bloqueo STV se aplica después del período de incubación 30 min. Como resultado, las partículas virales en la superficie celular están obligados por STV dejando solamente los virus internalizados disponible para la tinción con STV-Cy3. La cantidad relativa de virus internalizado se puede calcular como la relación de virus internalizado (medido en el &#39;30 min + STV &#39;muestra) dividido por la cantidad total de virus (descrito por el &#39;30 min&#39; muestra). Como controles se sugiere incluir las células infectadas. La señal después de la tinción STV-Cy3 de células infectadas da el fondoresultante de el protocolo de tinción. Un control para la fijación IAV es el pre-tratamiento de las células con neuraminidasa bacteriana (NA). Escinde NA fuera de ácido siálico de las glicoproteínas celulares, eliminando de este modo los receptores de unión de la superficie celular. La azida de sodio (SA) es un potente inhibidor metabólico y por lo tanto bloquea la endocitosis 13. Las células tratadas con azida de sodio deben ser positivos para la unión virus pero negativo para la internalización.

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	<tbody>
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			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>41966-052</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-163</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>14190-169&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>VWR Calbiochem</td>
			<td>126579</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Life Technologies</td>
			<td>15630-100</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Sucrose</td>
			<td>Fluka</td>
			<td>84100</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS</td>
			<td>Biosolve BV</td>
			<td>20092391</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>3680</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-LINK NHS-SS-BIOTIN kit</td>
			<td>Fisher Scientific</td>
			<td><a href="https://extranet.fisher.co.uk/insight2_ch/getProduct.do?productCode=W9971E">W9971E&#160;</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Bio Rad Protein Bio Assay</td>
			<td>Bio Rad</td>
			<td>500-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>deepwell tubes (1.2 ml microtubes)</td>
			<td>Milian</td>
			<td>82 00 001</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>&#160;Lucerna chem&#160; Electron microscopy sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>ultracentrifuge tubes</td>
			<td>Hemotec HmbH</td>
			<td>253070</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fluka</td>
			<td>93420</td>
			<td></td>
		</tr>
		<tr>
			<td>STV-Cy3</td>
			<td>Life Technologies</td>
			<td>43-4315</td>
			<td></td>
		</tr>
		<tr>
			<td>STV</td>
			<td>Life Technologies</td>
			<td>43-4302</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium azide</td>
			<td>Fluka</td>
			<td>71290</td>
			<td></td>
		</tr>
		<tr>
			<td>bacterial neuraminidase/sialidase</td>
			<td>Sigma-Aldrich</td>
			<td>N6514-1UN</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring attachment and internalization of influenza a virus in a549 cells by flow cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

Un dibujo animado que describe las cuatro condiciones experimentales diferentes se muestra en la Figura 1 Los resultados de un experimento representativo se presentan en las Figuras 2 -. 5. En la muestra &quot;0 min&quot;, virus biotinilado es frío unida a células que pueden ser visualizadas por tinción STV-Cy3 objetivo (Figuras 1 y 2). Cuando se aplica una etapa de bloqueo (en el &quot;0 min + STV&quot; muestra), el...

Watch this Scientific Journal Video about Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry at JoVE.com

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

Medición de apego y Internalización del Virus de la Influenza en células A549 por Citometría de Flujo

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

measuring-attachment-internalization-influenza-virus-a549-cells-flow

Research

JoVE Journal

Immunology and Infection

9.9K vistas.  University of Zurich. We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry. 

Video: Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Medición de la actividad calpaína en células fijadas y vivir por citometría de flujo

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Investigación

Inmunología e Infección

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

El examen de selección tímica positivo y negativo por citometría de flujo

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Propagación del virus de la influenza en embrionados de pollo Huevos

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

In Vitro El desmontaje de virus de influenza A cápsides por centrifugación en gradiente

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Estudios de Virus de influenza A en un modelo murino de infección

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Medición de Influenza neutralizando las respuestas del anticuerpo al virus a (H3N2) en suero humano por microneutralización ensayos utilizando células de MDCK-SIAT1

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Aislamiento y cultivo In Vitro de los macrófagos alveolares murinos y humanos

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

Preparación de la médula ósea entera para el análisis de citometría de masa de células de linaje neutrófilo

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Medición de la fagocitosis de Aspergillus fumigatus Conidia por leucocitos humanos utilizando citometría de flujo

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.