This work was supported by the Swedish Cancer Society (11 0253, 13 0469), the Swedish Research Council (K2012-99X-21959-01-3), Marcus Borgstr&#246;ms Foundation and the Swedish Research Council through a grant to the Uppsala RNA Research Centre (2006-5038-36531-16). We are indebted to the BioVis core facility at Uppsala University for much help with the FACS analysis.

El protocolo describe el aislamiento de células linfoides de material de paciente humano y por lo tanto requiere una aprobación ética. El trabajo realizado en el presente estudio fue concedida por el Comité de Revisión Ética de Uppsala (. Dnr 2013/387). 1. Aislamiento de células mononucleares (CMN) desde Humanos Palatine amígdalas PRECAUCIÓN: Todo el material no apantallado de origen humano como la sangre, tejidos o fluidos corporales debe considerarse como material potencialmente infectado. Por lo tanto, se deben seguir las prácticas de bioseguridad recomendadas para el manejo de los tejidos humanos. Nota: Para evitar la contaminación, todas las soluciones y equipos de cultivo de células deben ser estériles. Todos los tampones y soluciones deben ser pre-enfriados y se mantuvieron en hielo. Tejidos de amígdalas y células aisladas se deben mantener y manipulados en hielo. <ol><li> Obtener amígdalas frescos de los pacientes sometidos a amigdalectomía o amigdalotomía (Figura 2). Sumérgete la Clum tejidops en un tubo de centrífuga de 50 ml lleno de solución salina enfriada con hielo estéril Hanks equilibrada (HBSS) suplementado con suero bovino fetal 5% (FBS), mM glutamina 10, 0,05 mg / ml de gentamicina y 1% mezcla de antibiótico-antimicótico (penicilina, La estreptomicina y anfotericina B). </li><li> Mantenga los tubos con las muestras de tejido de las amígdalas sumergidas en todo momento en el hielo. Trate de procesar las amígdalas tan pronto como sea posible, y no más tarde de 3 horas después de la extirpación quirúrgica. </li><li> Coloque la amígdala en una placa de cultivo celular de plástico de 60 mm en el hielo y mantener el tejido humedecido con HBSS. Retire los coágulos sanguíneos visibles, grasa y tejido conectivo con un limpio fórceps de la superficie de las amígdalas. </li><li> Utilice una nueva placa de cultivo celular de 60 mm que contiene 5 ml de HBSS. Cortar el grupo tejido de las amígdalas en fragmentos 3-10 mm utilizando un par de tijeras estériles y / o un escalpelo. </li><li> Prepare una nueva placa de cultivo celular de 60 mm que contiene 10 ml de HBSS y colocar un filtro de células de plástico de 100 micras en la solución de HBSS. </li><li> Transfiera los dissfragmentos de amígdalas ejadas en el filtro de células con unas pinzas esterilizadas. Usando el extremo del émbolo de una jeringa de plástico, sin problemas exprimir los fragmentos de tejido a través del filtro de células. Asegúrese de que los fragmentos de amígdalas están totalmente inmersos en la HBSS. </li><li> Desechar el filtro de células con los restos de tejido y transferir la suspensión de células resultante de la placa de cultivo celular 60 mm en un tubo de centrífuga de plástico de 50 ml. Deje el tubo en hielo mientras se procede con los pasos 1,8 y 1,9. </li><li> En el caso de grandes amígdalas, de más de 2 cm de tamaño (Figura 2), apretar los fragmentos de la amígdala en dos coladores celulares para evitar la obstrucción de la malla en el colador. </li><li> Obtener el volumen final de 35 ml de la suspensión celular. Nota: En este paso es posible preparar la suspensión de células para la crioconservación (ver sección 2). </li><li> Añadir 10 ml de solución de gradiente de densidad de Ficoll como en un nuevo tubo de centrífuga de 50 ml. Superponer suavemente los suspens celularesion (paso 1.9) en la parte superior de la solución de gradiente de densidad. Evitar la mezcla de la suspensión de células y la solución por gradiente de densidad. Nota: La solución de gradiente de densidad tiene que ser calentado a la temperatura ambiente. </li><li> Centrifugar la suspensión de células en un rotor oscilante a cabo a 700 xg durante 20 min a TA. No active la función de freno de la centrífuga como el frenado rápido puede perturbar el gradiente. Asimismo, el uso de la función de aceleración más bajo disponible en la centrífuga. Nota: Después de este paso de centrifugación, observe MNCs como una capa esponjoso blanco en la interfase, y los glóbulos rojos (GR), los fibroblastos y los restos celulares como el sedimento en el fondo del tubo. </li><li> Recoger cuidadosamente la capa multinacionales utilizando una pipeta de 10 ml. Coloque la suspensión de células en un nuevo tubo de centrífuga de 50 ml. </li><li> Añadir 25 ml de helado de PBS a la suspensión celular y hacer girar el tubo a 300 xg durante 5 min en un rotor basculante a 4 ° C. </li><li> Eliminar el sobrenadante usando una pipeta de 25 mly repetir sedimento celular lavado paso dos veces más. Finalmente resuspender el botón celular en 15 ml de PBS. Nota: Después de la etapa de lavado final, es posible criopreservar la suspensión de células (véase la sección 2). </li><li> Aplicar 10 l de suspensión celular en la celda hemocitómetro cámara de recuento y contar el número de células bajo un microscopio. Dispensar la cantidad apropiada de células (por ejemplo, 2-3 de 7 x 10) en un tubo de centrífuga de 15 ml para la posterior separación de perlas magnéticas. Mantenga esta alícuota en hielo hasta el paso 3.2. </li></ol> 2. La criopreservación de células amigdalares <ol><li> Preparar medio de congelación (90% de SFB y 10% DMSO) y mantenerlo en hielo. Para el almacenamiento a largo plazo mantener a los medios de congelación a -20 ° C. </li><li> Determinar el número total de células utilizando una cámara de recuento celular hemocitómetro (paso 1,15). Calcular la cantidad necesaria de medio de congelación de acuerdo con la densidad celular congelado deseado (por ejemplo, 10 7 célulass / ml de medio de congelación). </li><li> Centrifugar la suspensión de células a 300 xg durante 5 min. Decantar el sobrenadante sin alterar el sedimento celular y resuspender el sedimento celular en medio de congelación enfriado con hielo (del paso 2.1). </li><li> Dispensar alícuotas de 1 ml de la suspensión celular en viales estériles diseñados para almacenamiento a largo plazo en nitrógeno líquido. Congele los viales en una cámara de isopropanol y almacenar a -80 ° CO / N. Para la conservación a largo plazo la transferencia de los viales en un nitrógeno líquido que contiene el tanque de almacenamiento o una C congelador celular -140 °. </li><li> Para descongelar viales con células congeladas, calentarlas rápidamente en un 37 ° C baño de agua. Inmediatamente cuando se descongela, dispersar la suspensión celular en 10 ml de HBSS precalentado (con suplementos, paso 1,1) en un tubo de centrífuga de 15 ml. Girar el tubo a 300 xg durante 5 min, eliminar HBSS y resuspender el sedimento celular en la densidad celular deseada en HBSS (con suplementos, paso 1.1). Nota: La viabilidad del af multinacionalesdescongelación ter es de importancia, ya que la presencia de células muertas disminuirá el rendimiento final de B y los linfocitos T purificados. Nota: De acuerdo a nuestra experiencia, la viabilidad celular a menos de 80% reducirá la eficiencia de aislamiento de células. </li></ol> 3. Selección positiva de linfocitos T Población de amigdalares multinacionales Nota 1: Este protocolo se basa en la selección positiva de linfocitos humanos T CD3 + de MNCs amígdalas usando perlas magnéticas acopladas al anticuerpo CD3. Es posible iniciar esta sección de frescos (Sección 1) o los (Sección 2) las multinacionales congelados. Nota 2: Comience con 3 x 10 7 multinacionales. No exceda este número de células, ya que las columnas de separación pueden obstruir y esto reducirá la eficiencia de aislamiento. Utilice columnas más grandes si se manejarán más células. Los volúmenes utilizados en este protocolo han sido experimentalmente optimizado para el número de células con nosotrosed en nuestra configuración experimental. <ol><li> Preparar el tampón de separación [PBS (pH 7,2), 0,5% de albúmina de suero bovino (BSA) y EDTA 2 mM]. Filtrar el tampón de separación a través de un filtro de 0,45 micras y almacenar a 4 ° C. </li><li> Haga girar la suspensión multinacionales (paso 1,14) a 300 xg durante 5 min a 4 ° C. Resuspender el sedimento resultante de células en 240 l (80 l por cada 10 7 células) de tampón de separación enfriado con hielo. Transferir la suspensión celular en un tubo de 2 ml estéril. </li><li> Añadir 20 l de anticuerpo CD3 magnético a la solución de células. Incubar los tubos durante 1 hora a 4 ° C con mezclado suave continua para mantener las células en suspensión. </li><li> Transferir todo de la suspensión celular en un tubo de 15 ml que contiene 5 ml de tampón de separación enfriado con hielo para lavar las células. </li><li> Centrifugar el tubo a 300 xg durante 10 min a 4 ° C en un rotor basculante. </li><li> Mientras tanto configurar el separador y la columna magnética. Coloque el separador magnético para el soporte y colocar la columna en la SEPARator. Lavar la columna mediante la aplicación de 500 l de tampón de separación enfriado con hielo. Deseche el flujo a través. </li><li> Coloque un nuevo tubo de extracción de 15 ml por debajo de la columna. Mantenga el tubo de recogida en el hielo. </li><li> Descartar el sobrenadante (paso 3.5) mediante la pipeta y resuspender suavemente el sedimento celular en tampón de separación l 500. Aplicar la suspensión de células en la parte superior de la columna de pre-lavado y se deja correr a través. Nota: grupos de células pueden obstruir la columna y por lo tanto reducir la tasa de flujo. Para evitar este problema, coloque un filtro de células de plástico 40 micras en la parte superior de la columna. Pasando a través de las células esta malla será altamente mejorar la eficiencia de este método. </li><li> Lavar la columna 4 veces con 1,5 ml de tampón de separación (500 l para 10 7 células). Esperar a que el depósito de la columna a estar vacía (sin líquido debe ser observado en la columna) antes de aplicar la siguiente etapa de lavado. Nota: Obtener las células no marcadas CD3, considerados como fracción de linfocitos B, ya que soneluyó en el tubo de recogida. </li><li> Retire la columna del separador y el lugar en un nuevo tubo de extracción de 15 ml. Pipetear 2 ml de tampón de separación en la columna. Utilizando el émbolo suministrado con la columna eluir los linfocitos T seleccionados positivamente, que se consideran como la fracción de linfocitos T. </li><li> Determinar el número de células purificadas (paso 1,15) si es necesario. Las suspensiones celulares están ahora listos para experimentos posteriores. </li></ol> 4. Análisis de citometría de flujo aislada amigdalar linfocitos B y T PRECAUCIÓN: solución de paraformaldehído es irritante y carcinógeno. Use ropa protectora adecuada, guantes y protección para los ojos / la cara. Nota 1: Este protocolo describe el método para la tinción directa de las células T y B aislado por fluorescencia análisis de clasificación de células activadas (FACS). El propósito principal de este paso es evaluar la pureza de la célula aislada poblaciones after CD3 anticuerpo separación magnética. Para este propósito B establecido y marcadores de células T, los anticuerpos monoclonales CD20 conjugado con fluoróforo-FITC y CD2-APC, se utilizan. También la inclusión de anticuerpos de control de isotipo es muy recomendable para distinguir el &quot;fondo&quot; la unión no específica de los anticuerpos CD2 y CD20 (véase el paso 4.8). Nota 2: Es posible para mantener las fracciones de células purificadas en una solución de paraformaldehído al 1% (PFA) en la oscuridad a 4 ° C hasta el momento de tinción (por ejemplo, día siguiente,.). También el mismo procedimiento es aplicable si hay un intervalo de tiempo entre la tinción y análisis FACS. Recuerde que en ambos casos las células deben lavarse adecuadamente con PBSA (PBS que contenía 0,2% de BSA) tampón, antes de la tinción o análisis FACS. <ol><li> Saque 6 x 10 6 células de las multinacionales del paso 1,15 (antes de aplicar a la columna), fracción de linfocitos B desde el paso 3.9 y fracción de linfocitos T a partir del paso 3.10. </li><li> Haga girar la célulasuspensiones a 300 xg durante 5 min a 4 ° C utilizando un rotor basculante. </li><li> Eliminar el sobrenadante con pipeta y se lava el sedimento celular una vez con 1 ml de helado PBSA. Haga girar las suspensiones de células a 300 xg durante 5 min a 4 ° C. Eliminar el sobrenadante con pipeta y resuspender el células en 600 l de tampón PBSA helada. </li><li> Añadir 100 l de la suspensión celular a la parte inferior de un tubo de FACS. </li><li> Añadir 100 l de PBS que contiene 10% inactivado por calor suero humano a cada tubo, mezclar bien e incubar durante ~ 1 min a RT o 20 min en hielo. Nota: los linfocitos B llevan receptores Fc. Con el fin de bloquear la Fc de receptores de células purificadas las fracciones se incuban con 10% inactivado por calor suero humano. El suero humano es inactivado por calor mediante incubación a 56 ° C durante 1 hr. Divida el suero inactivado por calor en pequeñas alícuotas y almacenar congelados a -20 ° C. </li><li> Centrifugar la suspensión de células a 300 xg durante 5 min a 4 ° C en un swing de salida del rotor. </li><li> Eliminar el sobrenadante con pipeta y se lava el sedimento celular con 1 ml PBSA. Repetir la centrifugación (300 xg durante 5 min a 4 ° C). </li><li> Añadir 100 l PBSA en cada tubo en hielo. Añadir la cantidad apropiada de anticuerpo monoclonal conjugado con fluoróforo, de acuerdo con la recomendación del fabricante (por ejemplo, 20 l de anti-CD20 y / o 5 l de anticuerpos anti-CD2 en 100 l de PBSA). Nota: No se olvide de asignar tubos de control suficientes para el análisis FACS. En este protocolo los siguientes controles deben incluir: 1) células teñidas con los anticuerpos anti-CD2 y anti-CD20 de forma individual, 2) células teñidas con los anticuerpos anti-CD2 y anti-CD20 de control de isotipo de forma individual y, 3) células sin adición de anticuerpos. </li><li> Brevemente vórtice del tubo y se incuba durante 30 min a 4 ° C en la oscuridad. </li><li> Lavar 2 veces con PBSA. Añadir 2 ml de solución de PFA al 1% a las muestras. Deje que las células permanecen en la solución de PFA al 476; C en la oscuridad hasta el momento del análisis FACS. </li><li> Inmediatamente antes de ejecutar las muestras en la máquina de FACS, se lavan las células 2 veces con PBSA (300 xg durante 5 min a 4 ° C). Resuspender las muestras en 1 ml de PBS y mantienen a 4 ° C (o en hielo), protegido de la luz, antes de la separación en el citómetro de flujo. </li><li> Hacer la clasificación FACS siguiendo el protocolo del citómetro de flujo fabricante. </li></ol> 5. La detección por PCR de ADN Adenovirus en aislada amigdalar linfocitos B y T Nota 1: Los hexón de adenovirus cebadores de genes son AdRJC1 (5&#39;-GACATGACTTTCGAGGTCGATCCCATGGA-3 &#39;) y AdRJC2 (5&#39;-CCGGCTGAGAAGGGTGTGCGCAGGTA-3&#39;) 11. Estos cebadores generan un amplicón de 139 pb. El gen 18S rRNA anfitrión puede ser detectada con los cebadores tp206 (5&#39;-CCCCTCGATGCTCTTAGCTG-3 &#39;) y tp207 (5&#39;-TCGTCTTCGAACCTCCGACT-3&#39;). El tamaño de amplificación esperado es 300 pb. Nota 2: Cada PCRplazo incluye un control negativo (destilada H 2 O) y un control positivo de ADN obtenido a partir de la línea de células B humanas (BJAB) infectados con adenovirus humano tipo 5 12. <ol><li> Extraer ADN (de al menos 1 x 10 6 células, paso 3.11), utilizando el método de purificación de columna basada membrana de sílice. Extracto de ARN usando fenol y solución de isotiocianato de guanidina 13. </li><li> Añadir 100 ng de ADN de linfocitos B y T a una mezcla de reacción que contenía tampón 1x HF, 0,2 mM de mezcla de desoxinucleótido trifosfato (dNTP), 0,25 M de cada cebador y 1 U de alta fidelidad de ADN polimerasa en un volumen total de 20 l. </li><li> Realizar la amplificación por PCR bajo las siguientes condiciones de ciclo: 30 seg de desnaturalización a 98 ° C, seguido por 30 ciclos de 98 ° C durante 10 seg, 67 ° C (hexon) o 58 ° C (18S rRNA) durante 30 seg y 72 ° C durante 30 s. La reacción de PCR se terminó con una etapa de extensión final de 7 min a 72 ° C. </li><li> Separar el amplif PCR resultanteicación productos en un gel de agarosa al 1,5% en 1x tampón TBE y visualizar las bandas esperadas por tinción con colorante de ácidos nucleicos. </li></ol>

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The authors have nothing to disclose.

Uno de los factores más importantes que afectan a los resultados de este protocolo es el uso de material amigdalar fresco como el material de partida. Por lo tanto, las muestras de amígdalas deben procesarse dentro de 3 horas después de la cirugía. Las amígdalas pueden ser obtenidas de adultos y niños. El material amigdalar de los hijos es generalmente más pequeño debido a la extirpación quirúrgica parcial de las amígdalas (amigdalotomía). Por lo tanto, el número de MNCs obtiene a partir de muestras de amig...

Tonsils are collections of incompletely encapsulated lymphoid tissues that lie under, and in contact with, the epithelium in the upper aero-digestive tract. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. The paired palatine tonsils together with the nasopharyngeal tonsil (adenoid), paired tubal tonsils and lingual tonsils constitute the so-called &#8220;Waldeyer&#180;s ring&#8221;. The latter is responsible for the initial contact between inhaled or ingested pathogens and the lymphoid tissues of the aerodigestive tract1,2. Indeed, numerous reports have shown that both bacterial and viral antigens can be detected in palatine tonsil tissue samples2-6.
The palatine tonsils are composed of dense lymphoid tissue covered by a stratified squamous non-keratinising epithelium. The tonsils have numerous crypts, epithelial invaginations, which penetrate the parenchyma increasing the surface area. Histologically, the palatine tonsils contain numerous lymphoid follicles with germinal centers, which are the sites for B cell maturation and differentiation (B-cell areas). Likewise, the palatine tonsils encompass T cells, which are mainly located in the extrafollicular regions (T-cell areas). In addition to the B and T cells, also various follicular dendritic cells can be detected in palatine tonsils1,2. 
Due to their anatomic location, the palatine tonsils are easily accessible by surgical interventions. For example, surgical removal of tonsils, known as tonsillectomy, is routinely carried out worldwide7. In children with tonsillar hyperplasia, a partial surgical removal of the tonsils (tonsillotomy) is sometimes used, causing less postoperative pain to the patients. Considering the accumulation of various pathogens in tonsils, surgically removed tonsils provide a unique opportunity to study the influence of viral and bacterial agents on tonsillar lymphocyte functions2,8. Furthermore it is possible to study if some pathogens prefer to reside in specific cell subpopulations9. In addition, as the tonsils are rich source of B lymphocytes, isolated tonsillar B lymphocytes can be efficiently used to study the activity of different B cell subpopulations10. However, as the palatine tonsils contain a mixture of cell types an efficient method to separate the different cell subpopulations is needed. 
Here, we describe a simple method for efficient and rapid isolation of tonsillar B and T cell populations from human palatine tonsils by using a magnetic-activated cell separation technique (Figure 1). The method described here is useful for scientists who want to assess the role of different infectious agents in human lymphoid organs such as palatine tonsils.

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			<td>Hanks balanced salt solution (HBSS)&#160;</td>
			<td>Gibco</td>
			<td>14175-053</td>
			<td>Contains 5% fetal bovine serum (FBS), 10 mM Glutamine,&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>&#160;0.05 mg/ml Gentamicin and 1% Antibiotic-Antimycotic mix</td>
		</tr>
		<tr>
			<td>Freezing medium</td>
			<td></td>
			<td></td>
			<td>90% FBS and 10% DMSO</td>
		</tr>
		<tr>
			<td>MACS buffer</td>
			<td></td>
			<td></td>
			<td>PBS (pH 7.2), 0.5% BSA and 2mM EDTA</td>
		</tr>
		<tr>
			<td>PBSA&#160;</td>
			<td></td>
			<td></td>
			<td>PBS containing 0.2% BSA</td>
		</tr>
		<tr>
			<td>PFA</td>
			<td></td>
			<td></td>
			<td>PBS containing 1% paraformaldehyde (PFA). Make fresh.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>PFA is suspected carcinogen. Wear gloves and goggles.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic mix</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>100-mm petridish</td>
			<td>Nunc</td>
			<td>172958</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting foreceps</td>
			<td>Fisher Scientific</td>
			<td>1381241</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight iris scissors&#160;</td>
			<td>Fisher Scientific</td>
			<td>12912055</td>
			<td></td>
		</tr>
		<tr>
			<td>disposable scalpels</td>
			<td>Swann-Morton</td>
			<td>REF 0501</td>
			<td></td>
		</tr>
		<tr>
			<td>100&#956;m plastic cell strainer</td>
			<td>Corning Life Sciences</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>40&#956;m plastic cell strainers&#160;</td>
			<td>Corning Life Sciences</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>2-ml plastic syringe</td>
			<td>BD Biosciences&#160;</td>
			<td>300185</td>
			<td></td>
		</tr>
		<tr>
			<td>15-ml conical centrifuge tubes</td>
			<td>SARSTEDT</td>
			<td>62554502</td>
			<td></td>
		</tr>
		<tr>
			<td>50-ml conical centrifuge tubes</td>
			<td>SARSTEDT</td>
			<td>62547254</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-speed centrifuge with fixed-angle or swinging-bucket rotor</td>
			<td colspan="2">Thermo Scientific Heraeus Megafuge 16R</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll&#8211;Hypaque</td>
			<td>Sigma-Aldrich</td>
			<td>F5415-50ML</td>
			<td>Ficoll solution</td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Biological industries</td>
			<td>040071A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulphoxide (DMSO)</td>
			<td>SIGMA</td>
			<td colspan="2">D2650-5X5ML</td>
		</tr>
		<tr>
			<td>MACS MS columns&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS human CD3 MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-881</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS separator (Octo MACS)</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-109</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Merck Millipore</td>
			<td>1120180100</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>AnalaR NORMAPUR</td>
			<td>20302.293</td>
			<td></td>
		</tr>
		<tr>
			<td>CD2 conjugated to allophycocyanin (CD2-APC)&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>560642</td>
			<td></td>
		</tr>
		<tr>
			<td>CD20 conjugated to fluorescein isothiocyanate (CD20-FITC)&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>556632</td>
			<td></td>
		</tr>
		<tr>
			<td>Human serum&#160;</td>
			<td>Rockland Immunochemicals</td>
			<td>D119-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>Thermo Scientific</td>
			<td>F-530L</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>BD Falcon</td>
			<td>352003</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotube</td>
			<td>SARSTEDT</td>
			<td>72379</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSRII flowcytometer</td>
			<td>BD Biosciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva 4.1 software</td>
			<td>BD Biosciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleospin Blood&#160;</td>
			<td>Macherey-Nagel</td>
			<td>740951.50</td>
			<td>DNA isolation kit</td>
		</tr>
		<tr>
			<td>TRIzol&#160; reagent</td>
			<td>Life technologies</td>
			<td>15596</td>
			<td>RNA isolation reagent</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>The Paul Marienfeld GmbH &#38; Co. KG</td>
			<td>0610030</td>
			<td>cell counter device</td>
		</tr>
		<tr>
			<td>GelRed</td>
			<td>Biotium</td>
			<td align="right">41003</td>
			<td>Nucleic acid gel stain&#160;</td>
		</tr>
	</tbody>
</table>

efficient isolation protocol for b and t lymphocytes from human palatine tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Una separación eficiente de las multinacionales tonsilares resultados en subpoblaciones altamente purificadas de los linfocitos B y T. Esto fue confirmado por análisis de FACS usando anticuerpos anti-CD2 anti-CD20 y para detectar linfocitos B y T poblaciones, respectivamente (Figura 3A). Por el contrario, una separación ineficiente de MNCs resultados en las fracciones celulares que son una mezcla de células de ambas subpoblaciones de linfocitos B y T (Figura 3B). </...

Watch this Scientific Journal Video about Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils at JoVE.com

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Protocolo Aislamiento eficiente para B y linfocitos T a partir Humanos Palatine amígdalas

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

efficient-isolation-protocol-for-b-t-lymphocytes-from-human-palatine

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Immunology and Infection

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Upper Respiratory Tract Infections

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15.7K vistas.  Uppsala Biomedical Center, Uppsala University. Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Video: Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Aislamiento de linfocitos T humanos, cultura y análisis de la migración In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Investigación

Inmunología e Infección

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

La expansión de los linfocitos T citotóxicos sangre del cordón umbilical que Citomegalovirus Target, Virus de Epstein-Barr, adenovirus y

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Aislamiento de linfocitos de ratón del tracto genital Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Aislamiento de los precursores de células B subconjuntos de Sangre del Cordón Umbilical

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Aislamiento y diferenciación de Th17 Naïve linfocitos T CD4

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

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 ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

La transfección altamente eficiente de humanos THP-1 macrófagos por Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

Un protocolo simple y rápida para no enzimáticamente disociarse Tejidos Humanos frescas para el Análisis de los linfocitos infiltrantes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Generación de recombinante humano IgG anticuerpos monoclonales a partir de células inmortalizadas B Ordenada

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

&lt;em&gt;In Vitro&lt;/em&gt; Differentiation of Human CD4&lt;sup&gt;+&lt;/sup&gt;FOXP3&lt;sup&gt;+&lt;/sup&gt; Induced Regulatory T Cells (iTregs) from Na&amp;#239;ve CD4&lt;sup&gt;+&lt;/sup&gt; T Cells Using a TGF-&amp;#946;-containing Protocol

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

Diferenciación in vitro de las células CD4 + humano FOXP3 + células T reguladoras (Induced iTregs) de T naive CD4 + células utilizando un protocolo de TGF-β que contienen