El estudio es apoyado por el Ministerio de Educación, Singapur (ACRF Tier1-RG40 / 13 y Tier2-MOE2013-T2-2-038). El manuscrito fue editado por Amy Sullivan de Obrizus Comunicaciones.

Todos los ratones son criados y mantenidos en condiciones libres de patógenos específicos y todos los protocolos de ratón se llevan a cabo de acuerdo con las directrices del Comité para el Cuidado y Uso de Animales institucional. 1. Preparación de tampones y reactivos <ol><li> Preparar Memorial Institute completa Roswell Park (RPMI) medio (10% de suero inactivado por calor bovino fetal (FBS), 2 mM L-glutamina, penicilina (100 UI / ml) / estreptomicina (100 mg / ml), 55 mM 2-mercaptoetanol ). </li><li> Preparar 20x solución salina equilibrada (BSS) Lote 1 y 2, por separado. <ol><li> Preparar 20x BSS stock 1 (111 mM dextrosa, fosfato de potasio 8,8 mM, 26,7 mM de fosfato dibásico de sodio en 1 L de agua estéril) y añadir 40 ml de 0,5% de rojo fenol 20x BSS stock 1 antes del ajuste del volumen final. Filtro estéril utilizando 0,2 micras filtro antes de guardar a 4 ° C. </li><li> Preparar 20x BSS madre 2 (25,8 mM de cloruro de calcio dihidrato, 107 mM de cloruro de potasio, cap 2,73 M de sodioLoride, 19,6 mM de cloruro de magnesio hexahidratado, 16,6 mM de sulfato de magnesio en 1 L de agua estéril). Filtro estéril utilizando 0,2 micras filtro antes de guardar a 4 ° C. </li><li> Para preparar 1x BSS para uso experimental, diluir 50 ml de BSS stock 1 y 50 ml de BSS madre 2, por separado, en 400 ml de agua estéril cada uno. Combinar ambas soluciones diluidas, se ajusta a pH 7, y se añaden 20 ml de FBS (2%). Completar hasta 1 litro con agua esterilizada y el filtro estéril usando 0,2 micras filtro. NOTA: las soluciones madre del SRS se deben preparar por separado porque la mezcla de poblaciones concentradas directamente SRS podría dar lugar a la precipitación. </li></ol></li><li> De glóbulos rojos (RBC) Tampón de lisis <ol><li> Para preparar tampón de lisis RBC, la mezcla 9 partes de cloruro de stock de amonio (cloruro de amonio 155 mM en agua estéril) a 1 parte de la base Tris (Tris mM (hidroximetil) aminometano 130 en agua estéril, pH 7,65) antes de su uso. NOTA: las tiendas de soluciones madre estériles de cloruro de amonio y Tris-base a 4 ° C. Preparar lisis bUffer fresco para asegurar la lisis eficiente de los glóbulos rojos. </li></ol></li></ol> 2. Generación de linfocitos Suspensión de bazo o los ganglios linfáticos NOTA: Es importante preparar todos los reactivos y equipos necesarios para el experimento antes de la eutanasia y del ratón para generar suspensiones de células individuales de linfocitos tan pronto como sea posible para mantener la viabilidad celular por alto. <ol><li> La eutanasia experimental de ratón por dislocación cervical o asfixia con CO2. NOTA: A partir de este paso en adelante, todos los procedimientos experimentales deben llevarse a cabo de forma aséptica. </li><li> Sumergir todo el ratón en etanol al 70% antes de hacer ninguna incisión. Extirpar el bazo y los ganglios linfáticos de manera aséptica 8, y colocarlos en distintos tubos de 15 ml que contienen 5 ml de RPMI / FBS enfriado con hielo (RPMI con FBS al 2%) o BSS / FBS (del paso 1.2.3). NOTA: Como BSS impide eficiente lisis RBC, utilice RPMI para la preparación de la suspensión de células del bazo y cambiar a BSS faetlisis r RBC. </li><li> Para generar una suspensión de células de bazo o los ganglios linfáticos, coloque el órgano (s) entre dos piezas de malla de filtro de micras de células estéril 100 en una placa de Petri que contiene 2 ml enfriado en hielo RPMI / FBS o BSS / FBS. Usando el émbolo de una jeringa de 1 ml, triturar el órgano (s) hasta que se haya rasgado en partes muy finas. </li><li> Transferir la suspensión celular a un tubo de 15 ml y lavar la malla del filtro de células con helado de RPMI / FBS o BSS / FBS. Recoger la suspensión celular restante, añadirlo al mismo tubo de 15 ml y centrifugar a 453 g durante 5 min a 4 ° C. Eliminar el sobrenadante. NOTA: Vuelva a suspender las células sedimentadas colocando el tubo con los dedos antes de la adición de tampón de lisis de glóbulos rojos o medio en los pasos posteriores. </li><li> Preparar temperatura ambiente tampón de lisis (RT) RBC durante la centrifugación de la suspensión celular (véase la etapa 1.3.). Después de la granulación de las células y eliminar el sobrenadante, volver a suspender las células con 1 ml de tampón de lisis de glóbulos rojos por cada 10 8 células. Incubate la reacción de lisis a temperatura ambiente durante 3-4 min. </li><li> Deja de lisis de glóbulos rojos con 14 ml de helado de BSS / FBS y centrifugar a 453 g durante 5 min a 4 ° C. </li></ol> 3. Purificación de células B y T <ol><li> La purificación de las células B <ol><li> Contar las células utilizando un hemocitómetro. Vuelva a suspender hasta 10 8 células esplénicas en 300 l BSS / FBS y se añaden 50 l anti-CD43 microbolas magnéticas 9,10. Para eliminar las células muertas, añadir 30 l perlas magnéticas Anexina V. Incubar la suspensión de células en un refrigerador 4 ° C durante 30 min. </li></ol></li><li> La purificación de las células T <ol><li> Contar las células utilizando un hemocitómetro. Vuelva a suspender hasta 10 8 células en no-T cóctel de anticuerpos agotamiento de las células (anticuerpos biotinilados contra CD19, B220, Gr-1, TCR-γδ, CD49b, CD11c, CD11b, Ter119 y CD4 o CD8 en función de la población de células diana a ser purificado), diluido 1: 200 en 200 BSS l / FBS 4,5. Incubar la suspensión de células en una4 ° C refrigerador durante 15 min. </li><li> Después de la incubación, añadir 10 ml BSS / FBS para lavar las células y centrifugar a 453 xg durante 5 min a 4 ° C. Eliminar el sobrenadante y resuspender las células en 165 l BSS / FBS con 30 microperlas de estreptavidina l y 15 l perlas magnéticas Anexina V. Incubar la suspensión de células en un refrigerador 4 ° C durante 30 min. NOTA: Para asegurar incluso el etiquetado con las microperlas magnéticas, incubar las células con microperlas de 15 min, a continuación, mezclar la suspensión de células suavemente tocando el tubo de 15 ml y se incuba otros 15 minutos durante la etapa 3.1.1. o 3.2.2. </li></ol></li><li> Preparación de la columna de separación para la purificación de la célula <ol><li> Preparar una columna de separación no utilizado durante el etiquetado de microperlas de las células (paso 3.1.1. O 3.2.2.). Pre-caliente BSS (sin FBS) a TA y utilizar 2 ml de lavar y equilibrar la columna de forma aséptica. Después de equilibrar con BSS, la columna se lava no se debe permitir que se seque. NOTA: Utilizamos el LS cOLUMNA lugar de la columna LD recomendada debido a su aptitud para la reutilización (véase el paso 3.4.). </li><li> Después del marcaje con las perlas magnéticas, añadir 14 ml BSS / FBS para lavar las células y centrifugar a 453 xg durante 5 min a 4 ° C. Eliminar el sobrenadante y resuspender las células en 1-3 ml de BSS RT. </li><li> Adjuntar una aguja estéril 21 G a la punta de la columna para reducir la velocidad de flujo durante el proceso de purificación. Cargar la suspensión celular en la columna equilibrada y se recoge el flujo a través que contiene las células diana purificadas. NOTA: Evitar la introducción de burbujas dentro de la columna durante la carga. </li><li> Lavar la columna una vez con 1 ml de BSS / FBS y se recoge el flujo a través que contiene las células diana purificadas. Actualizar la columna con el flujo a través una vez más. Recoger el flujo a través de después de la segunda carga en el mismo tubo de 15 ml. </li><li> Lavar la columna 3 veces con 1 ml de BSS / FBS y se recoge el flujo a través que contiene las células diana purificadas. A partir de entonces, se añaden 5 ml de BSS / FBS a thcolumna de dirección y, con un émbolo, a eliminar las células marcadas magnéticamente fuera de la columna en un nuevo tubo de 15 ml. </li><li> Comprobar la pureza de las células recogidas por citometría de flujo utilizando anticuerpos que se unen a antígenos de B purificada o células T 4,5 superficie. </li></ol></li><li> La reutilización de la columna de separación NOTA: La columna LS se puede reutilizar hasta 4 veces sin afectar a la eficiencia de purificación. <ol><li> Lavar la columna 3 veces con 5 ml de tampón fosfato salino (PBS) y 3 veces con 5 ml de agua destilada por la parte superior mediante el émbolo. </li><li> Lavar la columna con 5 ml de etanol 70% y se seca la columna extensivamente usando un grifo de aire para evitar la acumulación de óxido en la columna. </li><li> Para preparar una columna LS usado para un experimento de purificación por separado, lavar la columna de abajo hacia arriba con etanol 5 ml de 70% utilizando el adaptador de la jeringa. A continuación, lavar la columna de abajo hacia arriba dos veces con 5 ml de PBS estéril, seguido por 5 ml de PBS una vez desde la parte superior de la columna. Añadir 2RT ml de BSS para equilibrar la columna y luego proceder a la carga de la columna con células marcadas. </li></ol></li></ol> 4. CFSE etiquetado y Estimulación NOTA: Purificado células se pueden someter a una variedad de in vitro y en ensayos funcionales in vivo. Aquí, nosotros usamos las células T purificadas para determinar la capacidad de estimulación de células T de APC 5. <ol><li> solución de pre-calentamiento de etiquetado (0,1% de FBS en PBS) a 37 ° C antes de la CFSE de carga. NOTA: El uso de un bajo porcentaje de FBS en PBS reduce la muerte celular durante la CFSE carga y reduce al mínimo la pérdida de células durante la centrifugación. Sin embargo, demasiado FBS pueden interferir con la carga CFSE. </li><li> Lavar las células purificadas dos veces con solución de etiquetado, a continuación, se resuspendieron a 2 x 10 7 células / ml en solución de etiquetado pre-calentado en un tubo de 15 ml. </li><li> Preparar 10 solución CFSE mu M (1: 500 dilución de CFSE mM solución madre 5) en solución de etiquetado pre-calentado. CFSE solución debedebe prepararse cada vez para conseguir el marcaje óptimo. </li><li> Para cargar las células con CFSE, añadir la suspensión de células de 1 parte a 1 M solución CFSE parte 10 en un tubo de 15 ml y se incuban en la oscuridad durante 10 min a 37 ° C. Una concentración final de 5 mM CFSE se utiliza para etiquetar 1 x 10 7 células / ml. </li><li> Invertir el tubo cada 2 minutos para asegurar una mezcla homogénea de las células durante la CFSE de carga. </li><li> Para detener la reacción, añade varios volúmenes de medio RPMI completo enfriado con hielo y centrifugar a 453 g durante 5 min a 4 ° C. Al éxito CFSE de carga, el sedimento celular aparecerá amarillento. </li><li> Wash CFSE cargado células una vez más con medio RPMI completo enfriado con hielo y girar hacia abajo a 453 xg durante 5 min a 4 ° C antes de usar para el cultivo in vitro o la estimulación in vivo. </li></ol> 5. estimulación in vitro <ol><li> Preparar una solución 2x ​​social de estímulos (2x estímulos solución madre) inmediatamente antes de su uso para que 100 l de solución de los estímulos de la 2x se puede añadir a 100 l de células a un volumen final de 200 l por pocillo en una placa de 96 pocillos. </li><li> Si se requiere una placa recubierta con estímulos (IgM o CD40 para células B o CD3 y CD28 para las células T), diluir los estímulos en PBS y pre-capa de la placa de cultivo a 4 ° C durante la noche. Alternativamente, la placa de cultivo se puede recubrir a 37 ° C durante 1 hr en el día del experimento. Lavar la placa recubierta dos veces con PBS (No permita que la placa se seque en cualquier momento). </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Para las células B </td><td></td></tr><tr><td> Los estímulos </td><td> La concentración final </td></tr><tr><td> F (ab &#39;) 2 de cabra anti-IgM de ratón </td><td> 0,6 a 2,4 g / ml </td></tr><tr><td> Anti-ratón CD40 </td><td> 0,5-2 mg / ml </td></tr><tr><td> recombinanteratón IL-4 </td><td> 25 U / ml </td></tr><tr><td> lipopolisacárido </td><td rowspan="2"> 0,1 a 10 mg / ml </td></tr><tr><td> (LPS) de E. coli serotipo 055: B5 </td></tr><tr><td></td><td></td></tr><tr><td> Para las células T </td><td></td></tr><tr><td> Los estímulos </td><td> La concentración final </td></tr><tr><td> Anti-CD3 (placa recubierta) </td><td> 2-10 g / ml </td></tr><tr><td> (50 l / pocillo para el recubrimiento) </td><td></td></tr><tr><td> Anti-CD28 (placa recubierta) </td><td> 2 g / ml </td></tr><tr><td> Recombinante IL-2 </td><td> 40 U / ml </td></tr><tr><td> PDBu (éster de forbol) </td><td> 5-50 ng / ml </td></tr><tr><td> A23187 (ionóforo de calcio) </td><td> 250 ng / ml </td></tr></tbody></table> Tabla 1: Concentraciones de estímulos que se utilizan para estimular los linfocitos en cultivo in vitro.<ol start="3"><li> Para las células B marcadas con CFSE, vuelva a suspender a 3 x 10 6 células / ml en medio RPMI completo y la cultura, por triplicado, con 3 x 10 5 células / pocillo en placas de 96 pocillos de fondo plano de 72 horas. </li><li> Para las células T marcadas con CFSE, volver a suspender a 0,5-3 x 10 6 células / ml en medio RPMI completo y la cultura por triplicado con 0,5-3 x 10 5 células / pocillo en placas de 96 pocillos de fondo redondo de 48 o 72 horas . </li></ol> 6. En Vivo Estimulación <ol><li> Para la estimulación in vivo, la transferencia adoptiva de 4 x 10 6 células T marcadas con CFSE por ratón (por vía intravenosa (iv) en 200 l de PBS) en cada ratón receptor emparejado-MHC. NOTA: En este protocolo, las células T marcadas con CFSE se pueden transferir de manera adoptiva utilizando vena de la cola o inyección retro-orbital que estas células se alberga órganos linfoides como el bazo y los ganglios. </li><li> Desafiar a los ratones receptores un día después con el antígeno. NOTA:En este ejemplo, se utiliza la ovoalbúmina (proteína OVA, 50 g / ratón) como el antígeno porque específica de OVA, receptor de células T (TCR) las células T -transgenic se transfirieron adoptivamente en ratones receptores. Preparar la proteína OVA en PBS estéril e inyectar 100 l de proteína OVA / PBS o PBS control, a través de inyección subcutánea (IS), en cada ratón receptor 5. </li><li> Cosecha y generar suspensiones individuales de células de los órganos linfoides (ganglios linfáticos y el bazo) de los ratones receptores de 3 días después de la inmunización con la proteína OVA o PBS. ganglios linfáticos separadas en los ganglios linfáticos proximales (PLN), que incluye axilar, braquial y de los ganglios linfáticos cervicales superficiales) y los ganglios linfáticos distales (DLN), que incluye mesentérica, poplítea, inguinal, lumbar, y los ganglios linfáticos caudal. células de tinción de FACS utilizando los anticuerpos adecuados para detectar la proliferación de células T. NOTA: En este experimento de seguimiento de células CFSE, CFSE marcado con las células T de ratones de control inyectados con PBS establecer una fluorescencia de línea de base para los no-diViding células. Divisiones celulares de proliferación, las células, marcadas con CFSE estimulados por antígenos se visualizaron mediante la medición de los picos de fluorescencia 12,13. Con el control de PBS, el número de divisiones celulares de la proliferación, las células T marcadas con CFSE se puede determinar 12,13. </li><li> Analizar los datos de proliferación de células CFSE marcado mediante la comparación del número de divisiones celulares o picos entre las muestras (Figuras 1 y 2). NOTA: Por ejemplo, marcado con CFSE, las células T específicos de OVA adoptivamente transferidas en ratones receptores que recibieron el antígeno OVA se someterán a la proliferación activa en comparación con los ratones de control inyectados con PBS 5,12. Además, es digno de mención destacar que hay muchas maneras de analizar los datos de proliferación de células CFSE, como se demuestra por Hawkins y colegas 14. </li></ol>

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The authors have nothing to disclose.

En este protocolo, se demuestra un procedimiento para la purificación de los linfocitos de los órganos linfoides. la purificación de células utilizando la clasificación perla magnética es un método rápido y simple que produce células diana viables, altamente purificados. Los pasos críticos dentro del Protocolo La viabilidad celular y el rendimiento celular El mantenimiento de la viabilidad de las células del ...

Mature lymphocytes generally exist in the resting state if there is no pre-existing infection or inflammation in the individual. Therefore, it is important to retain the na&#239;ve status of lymphocytes during the isolation process before performing in vitro or in vivo functional assays. The key to ensuring consistent and reproducible results is to limit any unnecessary manipulation of the cells. 
Magnetic cell sorting utilizes antibodies and microbeads to label cells so as to enrich the cell population of interest. With this approach, there are two purification strategies: positive enrichment and negative depletion. Positive enrichment enriches the cell population of interest using an antibody that binds to the target cells. Negative depletion, on the other hand, depletes non-target cells, leaving the cell population of interest. In our lab, we prefer negative depletion to positive enrichment because the binding of antibodies to the target cells could potentially alter cell features and behavior. In fact, many established cell surface markers suitable for the isolation of a particular cell population are also functional receptors. 
Magnetic cell sorting not only yields highly pure populations of viable target cells, it is also less time-consuming and avoids the cellular stress induced by high-pressure flow used in fluorescence-activated cell sorting (FACS). By labeling the unwanted cell populations and depleting them using a magnetic separation column, we are able to perform rapid cell isolation without compromising the viability of the target cell population. In this protocol, we demonstrate the use of negative depletion strategies to purify na&#239;ve B cells or T cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Materials</td>
		</tr>
		<tr>
			<td>RPMI 1640 (without L-Glutamine)</td>
			<td>Gibco</td>
			<td>31870025</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td></td>
			<td></td>
			<td>Heat inactivated&#160;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140114</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD43 magnetic microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-801</td>
			<td>Mix well prior use</td>
		</tr>
		<tr>
			<td>Streptavidin microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-101</td>
			<td>Mix well prior use</td>
		</tr>
		<tr>
			<td>Anti-Annexin V magnetic beads</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-201</td>
			<td>Mix well prior use</td>
		</tr>
		<tr>
			<td>MACS LD&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-901</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well U-bottom sterile culture plate</td>
			<td>Greiner Bio-one</td>
			<td>650180</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well F-bottom sterile culture plate</td>
			<td>Greiner Bio-one</td>
			<td>655180</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;m cell strainer mesh</td>
			<td></td>
			<td></td>
			<td>To sterilize using UV radiation prior use</td>
		</tr>
		<tr>
			<td>0.2 &#956;m&#160; sterile disposable filter units</td>
			<td>Nalgene</td>
			<td>567-0020</td>
			<td>Can be substituted with any sterile filter device</td>
		</tr>
		<tr>
			<td>CellTrace Violet</td>
			<td>Invitrogen</td>
			<td>C34557</td>
			<td>CTV for short; alternative to CFSE</td>
		</tr>
		<tr>
			<td>CellTrace Yellow</td>
			<td>Invitrogen</td>
			<td>C34567</td>
			<td>CTY for short; alternative to CFSE</td>
		</tr>
		<tr>
			<td>CellTrace Far Red</td>
			<td>Invitrogen</td>
			<td>C34564</td>
			<td>CTFR for short; alternative to CFSE</td>
		</tr>
		<tr>
			<td>Cell Proliferation Dye eFluor 670</td>
			<td>eBioscience</td>
			<td>65-0840</td>
			<td>CPD670 for short; alternative to CFSE</td>
		</tr>
		<tr>
			<td>PKH26</td>
			<td>Sigma Aldrich</td>
			<td>PKH26GL</td>
			<td>PKH26, alternative to CFSE</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Sigma Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma Aldrich</td>
			<td>S5136</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Sigma Aldrich</td>
			<td>P0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma Aldrich</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck Millipore</td>
			<td>S7653</td>
			<td>Can use from other sources</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma Aldrich</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma Aldrich</td>
			<td>M2643</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>Sigma Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma Aldrich&#160;</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>(5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)</td>
			<td>Molecular Probes</td>
			<td>C-1157</td>
			<td>Reconstitute in DMSO</td>
		</tr>
		<tr>
			<td>Phorbol 12,13-dibutyrate (PBDU, Phorbol ester)</td>
			<td>Sigma Aldrich</td>
			<td>P1269</td>
			<td></td>
		</tr>
		<tr>
			<td>A23187 (Calcium ionophore)</td>
			<td>Sigma Aldrich</td>
			<td>C7522</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies and recombinant protein</td>
		</tr>
		<tr>
			<td>CD11b biotin (clone m1/70)</td>
			<td>Biolegend</td>
			<td>101204</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD11c biotin (clone N418)</td>
			<td>Biolegend</td>
			<td>117304</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>Gr-1 biotin (clone RB6-8C5)</td>
			<td>Biolegend</td>
			<td>108404</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>Ter119 biotin (clone Ter119)</td>
			<td>Biolegend</td>
			<td>116204</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>TCR-&#947;&#948; biotin (clone GL-3)</td>
			<td>Biolegend</td>
			<td>118103</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD19 biotin (clone 6D5)</td>
			<td>Biolegend</td>
			<td>115504</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>B220 biotin (clone RA3-6B2)</td>
			<td>Biolegend</td>
			<td>103204</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD49b biotin (clone DX5)</td>
			<td>Biolegend</td>
			<td>108904</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD4 biotin (clone GK1.5)</td>
			<td>Biolegend</td>
			<td>100404</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD8 biotin (clone 53-6.7)</td>
			<td>Biolegend</td>
			<td>100704</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>F(ab&#8217;)2 goat anti-mouse IgM (plate coated)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>115-006-075</td>
			<td>50 &#181;l/well for coating (96-well)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD40 mAb (plate coated)</td>
			<td>Pharmingen&#160;</td>
			<td>553722</td>
			<td>50 &#181;l/well for coating (96-well)</td>
		</tr>
		<tr>
			<td>Recombinant IL-4</td>
			<td>ProSpec&#160;</td>
			<td>Cyt-282</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS from E. coli Serotype 055:B5</td>
			<td>Sigma Aldrich</td>
			<td>L-4005</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD3 (clone clone OKT3) (plate coated)</td>
			<td>eBioscience&#160;</td>
			<td>16-0037-85</td>
			<td>50 &#181;l/well for coating (96-well)</td>
		</tr>
		<tr>
			<td>Anti-CD28 (clone clone 37.51 ) (plate coated)&#160;</td>
			<td>eBioscience&#160;</td>
			<td>16-0281-85</td>
			<td>50 &#181;l/well for coating (96-well)</td>
		</tr>
		<tr>
			<td>Recombinant IL-2</td>
			<td>ProSpec</td>
			<td>Cyt-370</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin from chicken egg white, Ovalbumin</td>
			<td>Sigma Aldrich</td>
			<td>A7641</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and activation of murine lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

purificación celular magnética de linfocitos permite a los usuarios para purificar una población de células diana en un período relativamente corto de tiempo. Utilizando el protocolo de agotamiento, hemos sido capaces de aumentar el porcentaje de células T CD8 (OT-I en la recombinación de activación de gen-1 (RAG-1) ratones deficientes) de 72,8% (antes de la purificación) a 94,2% (después de la purificación; Figura 1 A) 4,5. Estos linfocitos purific...

Watch this Scientific Journal Video about Isolation and Activation of Murine Lymphocytes at JoVE.com

Isolation and Activation of Murine Lymphocytes

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Aislamiento y activación de los linfocitos murinos

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

isolation-and-activation-of-murine-lymphocytes

Research

JoVE Journal

Immunology and Infection

20.9K vistas.  Nanyang Technological University. Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Video: Isolation and Activation of Murine Lymphocytes

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Modelo murino de CD40-activación de las células B

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Investigación

Inmunología e Infección

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 ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Aislar y inmunoticción Los linfocitos y las células dendríticas de las placas de Peyer murinas de

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

El aislamiento, tratamiento y análisis de células gingivales murinos

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

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&#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 ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

Ensayo de adherencia estática para el Estudio de la integrina activación en linfocitos T

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

El aislamiento, la identificación y purificación de células epiteliales del timo murino

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Aislamiento de células estromales murinas en nódulos linfáticos

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

Aislamiento y Trasplante de diferentes Aged murinos tímicos injertos.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

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.

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 ...