Este trabajo fue apoyado por el NIEHS R01ES014470 (KMZ).

Antes de iniciar este protocolo, recoger ratones recién nacidos en los días postnatales 1-3 (P1-3) en un recipiente estéril anidado con ropa de cama caja original para protegerlo y abrigarlo. Es importante trabajar de manera rápida y eficiente a través de este protocolo para optimizar el rendimiento de la microglia. Por favor consulte la Tabla 1 para la lista completa de reactivos. 1. Elaboración de instrumentos, medios de cultivo, y platos <ol><li> Preparar 10 ml / cachorro microglía completo Media (MCM; 1x Esencial Mínimo de Medio Earle [MEM] suplementado con: 1 mM de L-glutamina, piruvato de sodio 1 mM, 0,6% v / v de D-(+)-glucosa, 100 g / ml La penicilina / estreptomicina (P / S), 4% v / v de suero bovino fetal (FBS), 6% v / v de suero de caballo), y añadir a cada matraz T-75 (1 matraz / PUP); tapa y horizontalmente en lugar una incubadora (37 ° C, 5% de CO 2) equilibrar durante 1 hora. Es esencial para equilibrar este medio en un matraz (s) antes de comenzar la disección. </li><li> PrepaRe, alícuota, y caliente en 37 ° C MCM baño de agua para la disección (6 ml / PUP) y en un tubo cónico de MCM separada para la resuspensión celular (2 ml / PUP). Preparar, alícuota, y enfriar en hielo Picado de medios (MM; HBSS suplementado con: 100 mg / ml P / S, 0,01 M HEPES; 3 ml / cría), y disección de medio (DM; MEM suplementado con 100 mg / ml P / S, 2 ml / perrito). </li><li> Esterilizar las siguientes herramientas quirúrgicas por inmersión en etanol al 70% durante 10 min: tijera # 1 (1; decapitar), fórceps # 1 (2, para asegurar la nariz y eliminar las cortezas), tijera # 2 (1; nonangled; para abrir el cuero cabelludo ), fórceps # 2 (1; para quitar el cuero cabelludo), fórceps # 3 (1; para eliminar cráneo), fórceps # 4 (2; para eliminar meninges), tijera # 3 (1; pelos en el tejido cerebral). Deje secar al aire en una gasa estéril. </li><li> Limpie la estación de trabajo y la disección microscopio horizontal con flujo de aire con 70% de etanol. </li><li> Dispense DM en 2, 100 mm placas de Petri estériles para la captura de la cabeza (3 ml; 1 plato / 3 cachorros) y la disección (1 ml;1 plato / crías; enfriada en hielo antes de la disección). Dispense MM en 1 estéril, 100 mm placa de Petri para picar el tejido cerebral (1 ml, 1 plato / perrito). </li></ol> 2. Tejido Cerebral Disección <ol><li> Antes de la eutanasia, desinfectar suavemente el cuello y la cabeza del cachorro con etanol al 70%, utilizando un pañuelo de papel de laboratorio. Con tijera # 1, la eutanasia rápidamente mediante la eliminación de las crías de la cabeza, la cabeza de captura en placa de Petri previamente preparado. La eutanasia y completar la disección de una cría antes de proceder a la siguiente animal. </li><li> Transfiera la cabeza al plato disección fría; bajo el microscopio de disección asegurar la cabeza apretando la nariz usando fórceps # 1 (como alternativa, la cabeza puede ser asegurada por la perforación de las pinzas a través de la cuenca del ojo); cuero cabelludo abierto utilizando tijeras # 2 por la creación de un Y-cut: comenzar desde la base de la cabeza y en un ángulo hacia los ojos. Utilice fórceps # 3 para eliminar suavemente el cuero cabelludo, quitando lateralmente, exponiendo el cráneo. </li><li> El uso de fórceps # 3, remover suavemente el tejido conectivo en la base decabeza. Cuando la base del cráneo está expuesto el cráneo, abra cuidadosamente enroscando un brazo de pinza entre el cerebro y el cráneo (siguiendo la cisura interhemisférica); agarre, tire suavemente el cráneo hacia arriba para rasgar. </li><li> Interrumpir el tejido conectivo debajo de la parte ventral de cerebro usando fórceps # 1, y posteriormente eliminar el cerebro utilizando la misma herramienta. Levantar el cerebro fuera del cráneo mediante el uso de una fuerza hacia arriba bajo la parte ventral del cerebro. </li><li> Mantener el cerebro en su posición anatómica: frente lado dorsal hacia arriba para visualizar las cortezas bajo el microscopio de disección; asegurar el cerebro en su lugar mediante la inserción de puntas de pinzas en la intersección del cerebro medio y la corteza; eliminar completamente las meninges corticales con fórceps # 4, a continuación, proceder a retirar las meninges ventral. Una vez que las meninges se han separado completamente, separe las cortezas de la parte restante del cerebro utilizando fórceps # 4. Es esencial para eliminar por completo el tejido meníngeo para maximizar la pureza microglia y yield. </li></ol> 3. Tejido Cerebral Picado <ol><li> Cortezas de transferencia para preparar previamente picado plato; seguir picando paso utilizando una técnica estéril en la categoría II del Gabinete de Seguridad Biológica. </li><li> Pique tejido cerebral usando tijera # 3, la transferencia de tejido picado a un tubo cónico de 15 ml;. Enjuague tijeras y picado plato con 1 ml de mM de cada uno, recoger y dispensar enjuagues en el tubo cónico de 15 ml que contiene el tejido picado Este paso de enjuague maximizará el rendimiento de la microglia, asegurando que todo el tejido residual se transfiere al tubo cónico. Deja suspensión de tejido en reposo durante 1-2 min, permitiendo tejido picado se asiente en el fondo del tubo. No exceda de 2 min como MM no contiene nutrientes esenciales. </li><li> Retire con cuidado y deseche 2 ml de sobrenadante con micropipeta genérica 1.000 l con una punta de pipeta estándar, asegurándose de no perturbar el tejido picado resuelto. </li&gt; <li> Añadir 3 ml de MCM a la-tejido picado; se tritura el tejido con toda su fuerza usando un 1,000 l genérico micropipeta con una punta de pipeta estándar No se discernible tejido sólido debe permanecer cuando la trituración es completa.. Agregar otro 3 ml de MCM con la misma punta de la pipeta, la recogida de la muestra se tritura residual de la punta de la pipeta. </li></ol> 4. El cultivo de células corticales <ol><li> Se centrifuga el tejido picado para que sedimenten las células (215 xg, 5 min; RT; centrífuga clínica con rotor basculante). Volver células sedimentadas a la cabina de seguridad biológica, retirar y desechar el sobrenadante, resuspender suavemente el sedimento celular (2 ml / tubo; MCM), añadir la resuspensión celular a la T-75 frasco previamente preparado; tapar el frasco y colocarla horizontalmente en un humidificado, incubadora de cultivo de tejidos estándar (37 ° C, 5% de CO 2). Permitir que las células se incuban durante 24 horas. </li><li> Después de 24 horas, toque suavemente frasco contra la palma de la mano para interrumpirrestos de tejido. Compruebe las células antes y después de tocar a asegurar la completa eliminación de los desechos. Frasco Set vertical; retirar y eliminar los medios de comunicación; añadir MCM fresco al fondo del frasco (12 ml; 37 ° C); tapa y colóquelo de nuevo en la incubadora, horizontalmente. </li><li> Compruebe células diaria de contaminación y para monitorear el crecimiento. </li><li> En aproximadamente 17 a 21 días in vitro (DIV), las células microgliales estarán listos para la cosecha del cultivo glial mixto. Para determinar si las células están listas, examinar las células bajo un microscopio de contraste de fase invertida. Microglia están listos para la cosecha, cuando en ~ 40% de confluencia. Aparecen como células pequeñas, brillante, esféricas, creciendo como una monocapa sobre otro glia. </li></ol> 5. El aislamiento de microglia de glial mixto Cultura <ol><li> Para aislar la microglía, toque vigorosamente frasco contra la mesa de laboratorio. Esta agitación ayuda a microglia separadas de otras células gliales debido a las propiedades de baja adherencia de la microglia. </li><li&gt; Sello T-75 caps matraz con Parafilm y rotar los cultivos mixtos gliales (200 rpm; 37 ° C, 5 h). utilizando un agitador rotatorio nonhumidified temperatura controlada No permitir que las células se dan durante más de 5 horas Inspeccione visualmente. que la microglia se han levantado de la superficie del matraz después de 5 horas, utilizando un microscopio de contraste de fase invertida. microglía son brillantes, esférico, células de flotación libre. Habrá una capa residual de astrocitos y oligodendrocitos que se adhieren a la parte inferior del matraz. </li><li> Recoger medio sobrenadante que contienen microglia en un tubo de centrífuga cónico estéril; microglia pellets (215 xg, 5 min; rotor con cubeta oscilante). </li><li> Resuspender el precipitado de la microglia en caliente Microglial Medio de Crecimiento (MGM; MEM suplementado con: 1 mM piruvato de sodio, 0,6% v / vglucose, 1 mM de L-glutamina, 100 mg / ml de P / S, 5% v / v de FBS; ~ 2 ml / matraz), determinar la concentración de células utilizando un hemocitómetro, y la placa de 1 x 10 5 células / pocillo en un formato de placa de 24 pocillos enplástico o resbalones estériles cubierta de vidrio (500 l / pocillo; MGM). Mediante este procedimiento el rendimiento promedio microglia es cortex 7,5 x 10 5 células / cachorro. </li><li> 24 horas después de la galvanoplastia, quite todos los medios de comunicación y las células de realimentación con MGM fresco (37 º C) para eliminar las células flotantes y escombros. Este paso es fundamental para la supervivencia celular y mantener microglia en reposo. Las células pueden ser utilizados para la experimentación inmediatamente después de la realimentación. </li></ol> 6. Tratamientos Ejemplo <ol><li> Exponer las células a 10 ng / ml de Pam 3 CSK 4 (Pam), 10 ng / ml de lipopolisacárido (LPS) o de control del vehículo; incubar durante 2 o 24 horas en incubador humidificado (37 ° C, 5% de CO 2). </li></ol> 7. Analizando Microglia para Funcionalidad ex vivo a través de inmunofluorescencia de Imágenes e inmunocitoquímica <ol><li> Después del tratamiento, los medios de cultivo celular de cosecha en tubos de microcentrífuga (1,5 ml) y Centrifuge utilizando una microcentrífuga de mesa (2 min; 900 xg) para borrar los medios de comunicación. Transferir el sobrenadante a un tubo de microcentrífuga limpio; ensayo inmediatamente o almacenar a -20 ° C hasta su utilización. Analizar la concentración de TNF-α en células de cultivo sobrenadante a través de kit de ELISA de TNF-α ratón comercialmente disponible. </li><li> Lavar las células con solución salina tamponada con fosfato (PBS), y fijar las células con 4% (w / v) de paraformaldehído / 4% (w / v) de sacarosa / PBS a pH 7,4 (4% de PFA; 15 min; RT). Después de la fijación, lavar las células con PBS durante 5 min con agitación suave en un agitador orbital. <ol><li> Inmunofluorescencia / inmunocitoquímica. Después de la fijación y lavado con PBS, la microglía proceso para inmunocitoquímica. Todos los pasos llevados a cabo con agitación suave. Permeabilizar las células en PBS/0.1% de Triton X-100; células de bloques en PBS/10% de suero normal de cabra durante una hora. Se incuban las células de O / N a 4 ° C con anticuerpo de conejo anti-NF-kB (subunidad p65; 1/1, 250) o de conejo anti-Iba-1 anticuerpo (1/750) en tampón de bloqueo. Visualize complejo antígeno: anticuerpo tras la incubación con fluorescente conjugado de cabra anti-conejo de anticuerpos IgG secundaria (1 hora, 1/1, 000). Se quita el anticuerpo secundario no unido por lavado con PBS/0.1% de Triton X-100 (3x 5 min). </li><li> Células de manchas contador con 4 &#39;,6-diamidino-2-fenilindol / PBS (DAPI; 0,1 g / ml). Para visualizar el núcleo de la célula Todos microglia derivados de CX3CR1-GFP + / - ratones expresan GFP. Utilizar un filtro de longitud de onda de 350 nm a visualizar DAPI; un filtro de longitud de onda de 488 nm a visualizar GFP, y un filtro de longitud de onda de 594 nm a visualizar p65 y Iba-1 inmunocomplejos. </li></ol></li><li> Monte la cubierta se desliza usando una solución de montaje acuoso y visualizar microglia utilizando microscopía fluorescente. </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murinemicroglial+cells+for+RNA+analysis+or+flow+cytometry.">Isolation of murinemicroglial cells for RNA analysis or flow cytometry.</a> Nat. Protoc. 1, 1947-1951 (2006).</li><li>Ford, A. L., Goodsall, A. L., Hickey, W. F., Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+adult+ramified+microglia+separated+from+other+central+nervous+system+macrophages+by+flow+cytometric+sorting.+Phenotypic+differences+defined+and+direct+ex+vivo+antigenpresentation+to+myelin+basic+protein-reactive+CD4++T+cells+compared.">Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigenpresentation to myelin basic protein-reactive CD4+ T cells compared.</a> J. Immunol. 154, 4309-4321 (1995).</li><li>Ford, A. L., Foulcher, E., Goodsall, A. L., Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+digestion+with+dispase+substantially+reduces+lymphocyte+and+macrophage+cell-surface+antigen+expression.">Tissue digestion with dispase substantially reduces lymphocyte and macrophage cell-surface antigen expression.</a> J. Immunol. Methods. 194, 71-75 (1996).</li><li>Pino, P. A., Cardona, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+brain+and+spinal+cord+mononuclear+cells+using+percoll+gradients.">Isolation of brain and spinal cord mononuclear cells using percoll gradients.</a> J. Vis. Exp. , (2011).</li><li>Veremeyko, T., Starossom, S. C., Weiner, H. L., Ponomarev, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+microRNAs+in+microglia+by+real-time+PCR+in+normal+CNS+and+during+neuroinflammation.">Detection of microRNAs in microglia by real-time PCR in normal CNS and during neuroinflammation.</a> J. Vis. Exp. , (2012).</li><li>Su, X., Federoff, H. J., Maguire-Zeiss, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+alpha-synuclein+overexpression+mediates+early+proinflammatory+activity.">Mutant alpha-synuclein overexpression mediates early proinflammatory activity.</a> Neurotox. Res. 16, 238-254 (2009).</li><li>Jung, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+fractalkine+receptorCX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptorCX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol. Cell. Biol. 20, 4106-4114 (2000).</li><li>Imai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+molecular+characterization+of+fractalkine+receptor+CX3CR1,+which+mediates+both+leukocyte+migration+and+adhesion.">Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion.</a> Cell. 91, 521-530 (1997).</li><li>Cardona, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+microglial+neurotoxicity+by+the+fractalkine+receptor.">Control of microglial neurotoxicity by the fractalkine receptor.</a> Nat. Neurosci. 9, 917-924 (2006).</li><li>Streit, W. J., Walter, S. A., Pennell, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+microgliosis.">Reactive microgliosis.</a> Prog. Neurobiol. 57, 563-581 (1999).</li><li>Frank, M. G., Wieseler-Frank, J. L., Watkins, L. R., Maier, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+highly+enriched+and+quiescent+microglia+from+adult+rat+hippocampus:immunophenotypic+and+functional+characteristics.">Rapid isolation of highly enriched and quiescent microglia from adult rat hippocampus:immunophenotypic and functional characteristics.</a> J. Neurosci. Methods. 151, 121-130 (2006).</li></ol>

Los autores declaran que la investigación se llevó a cabo en ausencia de cualquier relación comercial o financiero que puedan interpretarse como un posible conflicto de intereses.

El presente procedimiento ofrece un método eficaz para el aislamiento de la microglia corticales de ratones neonatales. Este procedimiento tiene un doble beneficio de 1) preservar inmunofenotipo la microglia y la funcionalidad, según lo determinado por proyección de imagen fluorescente, inmunocitoquímica, y ELISA, y, 2) que permite la microglía a madurar en presencia de otras células gliales (astrocitos y oligodentrocytes) antes de la aislamiento, lo que puede facilitar importantes interacciones célula-célula du...

Las células microgliales, los macrófagos de vigilancia de la parénquima del SNC, comprenden aproximadamente el 12% de la población total de células del cerebro de los mamíferos adultos. La microglía se derivan de saco vitelino mieloides precursoras células y varían en densidad celular y la morfología en diferentes regiones cytoarchitectural en el SNC adulto 1-5. En un cerebro adulto sano, la microglia son células pequeñas, ramificadas o polares, con procesos dinámicos finos. En contraste con la morfología de los macrófagos periféricos, microglia demuestran un reposo, de bajo perfil fenotipo en cerebros sanos que pueden aparecer como la inactividad celular, sin embargo, en los estudios de imagen in vivo demuestran que los procesos microgliales se extienden de forma dinámica y se retraen para monitorear su microambiente de una manera que recuerda a &quot; muestreo y de estudio &quot;6,7. La microglía son altamente y diferencialmente sensibles a alteraciones ambientales y fisiopatológicos en el cerebro, de conmutacióndesde un surveillant a un estados considerados como su reposo y activadas comúnmente estatales efectoras, respectivamente. Este interruptor de activación puede estar mediada por la participación de los receptores de membrana de reconocimiento de patrones (PRRS), como los receptores tipo Toll (TLR), que responden a patrones moleculares asociados a patógenos (PAMP), lipoproteínas de saber bacterianas-y derivados-viral , ácidos nucleicos, hidratos de carbono y 8-11. Además de PAMP, parentales también se han mostrado para inducir la activación microglial contra moléculas estériles, no patógenas conocidas como patrones moleculares peligro / daño-asociado (apaga), que representan una perturbación en la homeostasis del SNC, tales como daño celular 12-16. Una vez enganchado, PRRS inician una cascada de señalización intracelular que resulta en cambios en la morfología celular microglial y la expresión génica; específicamente, microglia activada adaptar un fenotipo-ameboide como, translocate la p65 de NF-kappa B subunidad (p65) para el núcleo de la célula, y la upregulate producción y la secreción de citocinas proinflamatorias, tales como factor de necrosis tumoral-α (TNF-α), la interleucina-1 β (IL-1β), junto con especies de oxígeno reactivas (ROS) 16-24. Aunque integral en la respuesta inmune innata del sistema nervioso central, también se han encontrado estas moléculas secretadas para aumentar el estrés oxidativo neuronal, induciendo de ese modo y exacerbando la neurodegeneración en estados de enfermedad tales como Parkinson y la enfermedad de Alzheimer 25-29. Sin embargo, los mecanismos de activación microglial en estados patológicos no se entienden completamente. Por lo tanto, el aislamiento de microglia es una poderosa herramienta de investigación en estos procesos biológicos, como muchos en funciones in vivo de la activación microglial puede ser recapitulado en la cultura. Varios métodos están disponibles para el aislamiento de la microglía, incluyendo el aislamiento a través de un gradiente de Percoll después de la digestión enzimática de tejido del SNC 30,31. Sin embargo, la digestión enzimática puede alterarel inmunofenotipo de las células mediante la reducción de la superficie celular la expresión del antígeno 32, y los resultados en el rendimiento de células por animal inferior que el método descrito en el presente documento. En concreto, se presenta un rendimiento promedio microglia por corteza cachorro de 7,5 x 10 5 células, mientras que los métodos de aislamiento de todo el SNC ya se ha informado a través de un rendimiento gradiente de Percoll 3-5 x 10 5 células 30,33,34. El presente procedimiento evita el uso de enzimas de digestión mediante el aislamiento de la microglia en base a sus propiedades de baja adherencia, preservando así el inmunofenotipo y la funcionalidad de la microglia. En el presente estudio, se describe el aislamiento de las células microgliales de mezcla de culturas gliales derivadas de heterocigotos CX3CR1-GFP (CX3CR1-GFP + / -) neonatales, y C57BL / 6 corticales murinas mediante agitación mecánica en un agitador rotatorio, una extensión de anterioridad publicado 24,35 método. Utilizamos la antigua cepa de ratón para facilitar la visualización de la microglia, comoestos ratones expresan GFP bajo el control del locus endógeno CX3CR1 - un promotor específico de los monocitos 36-38. Este método produce microglial culturas altamente puro con un inmunofenotipo conservados ex vivo como se demuestra por cambios morfológicos, la translocación nuclear de p65, y la secreción de TNF-α cuando son desafiados con lipopolisacárido bacteriano (LPS) o Pam 3 CSK 4, TLR4 y TLR1 / 2 agonistas , respectivamente.

<table border="0" cellpadding="0" cellspacing="0" width="680">
	<colgroup>
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="160">
			<td height="160" style="height:160px;width:233px;">Glucose</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:155px;">G8270</td>
			<td style="width:143px;">Make 20% Stock Solution with MilliQ water; filter sterilize; store at 4 &#176;C; shelf life: 3-6 months. Used to make MCM and MGM.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Sodium pyruvate 100 mM (100x)</td>
			<td style="width:149px;">Hyclone</td>
			<td style="width:155px;">SH30239.01</td>
			<td style="width:143px;">Store at 4 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Penicillin/Streptomycin 10,000 units/ml (100x)</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">15140-122</td>
			<td style="width:143px;">Store at -20 &#176;C. Used to make MCM, MGM, MM, and DM.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">L-glutamine 200 mM (100x)</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">25030-081</td>
			<td style="width:143px;">Store at -20 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Fetal Bovine Serum (Defined)</td>
			<td style="width:149px;">Hyclone</td>
			<td style="width:155px;">SH30070.03</td>
			<td style="width:143px;">Filter sterilize; store at -20 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:233px;">Minimum Essential Medium Earle&#39;s (MEM)</td>
			<td style="width:149px;">Cellgro</td>
			<td style="width:155px;">15-010-CV</td>
			<td style="width:143px;">Without L-glutamine. Contains Earle&#39;s salts. Used to make MCM, MGM, and DM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Horse Serum</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">16050</td>
			<td style="width:143px;">Filter sterilize; store at -20 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td style="width:149px;">Cellgro</td>
			<td style="width:155px;">21-021-CV</td>
			<td style="width:143px;">Without calcium and magnesium. Store at 4 &#176;C. Used to make MM.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">HEPES 1 M</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">15630-031</td>
			<td style="width:143px;">Store at 4 &#176;C. Used to make MM.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">T-75 Flask</td>
			<td style="width:149px;">Corning</td>
			<td style="width:155px;">430641</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">4&#39;,6-Diamidino-2-Phenylindole, dilactate (DAPI)</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:155px;">D3571</td>
			<td style="width:143px;">Used to stain cell nucleus.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Rabbit anti-Iba1</td>
			<td style="width:149px;">Wako</td>
			<td style="width:155px;">019-19741</td>
			<td style="width:143px;">Used at 1/750 dilution for ICC staining of Iba1.&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Rabbit anti-NF&#954;B (p65)</td>
			<td style="width:149px;">Abcam</td>
			<td style="width:155px;">7970</td>
			<td style="width:143px;">Used at 1/1250 dilution for ICC staining for p65.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:233px;">Alexafluor 594 Goat anti-Rabbit IgG (H+L)</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:155px;">A11012</td>
			<td style="width:143px;">Used at 1/1000 dilution for visualization of antigen:antibody complex in ICC.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">10 ml Disposable Serological Pipet</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">13-678-11E</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">50 ml Disposable Centrifuge Tube</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">05-539-8</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">15 ml Disposible Centrifuge Tube</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">05-539-12</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Sterile Polystyrene Petri Dish</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">875713</td>
			<td style="width:143px;">100 mm x 15 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Scissor: Straight Metzembaum (scissor #1)</td>
			<td style="width:149px;">Roboz Surgical</td>
			<td style="width:155px;">RS-6010</td>
			<td style="width:143px;">1; 5 inch; used for removing head</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Scissor: Vannas 
			(scissor #2)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">15000-08</td>
			<td style="width:143px;">1; non-angled; 2.5mm cutting edge; used to open scalp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Scissor: Student Vannas (scissor #3)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">91501-09</td>
			<td style="width:143px;">1; curved; used to mince brain tissue</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Forcep: Dumont #7 
			(forcep #1)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">91197-00</td>
			<td style="width:143px;">2; used to secure nose and remove cortices&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Forcep: Dumont #2 
			(forcep #2)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">11223-20</td>
			<td style="width:143px;">1; used to remove scalp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Forcep: Dumont #3 
			(forcep #3)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">11231-30</td>
			<td style="width:143px;">1; used to remove skull</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Forcep: Dumont #5a 
			(forcep #4)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">11253-21</td>
			<td style="width:143px;">1; used to remove meninges</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Table of specific equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Name of Equipment</td>
			<td style="width:149px;">Name of Company</td>
			<td style="width:155px;">Catalogue Number</td>
			<td style="width:143px;">Comments</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Zoom Stereo Dissection Microscope&#160;</td>
			<td style="width:149px;">Olympus</td>
			<td style="width:155px;">SZ4060</td>
			<td style="width:143px;">Microscope is placed inside Laminar-Horizontal Flow Cabinet</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Laminar-Horizontal Flow Cabinet</td>
			<td style="width:149px;">Nuaire</td>
			<td style="width:155px;">NU-201-330</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Biological Safety Cabinet</td>
			<td style="width:149px;">Labconco</td>
			<td style="width:155px;">3440001</td>
			<td style="width:143px;">Class II</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:233px;">Water-Jacketed CO2 Incubator</td>
			<td style="width:149px;">VWR</td>
			<td>97025-836</td>
			<td style="width:143px;">Set to 37 &#176;C, 5% CO2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Swing-out buckets</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>75006441</td>
			<td style="width:143px;">To be used with Swing-out rotor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Swing-out Rotor</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>75006445</td>
			<td style="width:143px;">Max Radius: 19.2 (cm)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Sorvall Legend RT+ Centrifuge 
			(clinical centrifuge)</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>75-004-377</td>
			<td style="width:143px;">With swing-out rotor</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">AccuSpin Micro 17 microcentrifuge 
			(tabletop microcentrifuge)</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>S98645</td>
			<td style="width:143px;">With microliter rotor (24 x 1.5/2.0 ml; 
			Cat #: 75003524)&#160;&#160;</td>
		</tr>
	</tbody>
</table>

isolation of cortical microglia with preserved immunophenotype and functionality from murine neonates

El aislamiento de microglia de tejido del SNC es una poderosa herramienta de investigación utilizada para estudiar la biología microglial ex vivo. El presente método detalla un procedimiento para el aislamiento de la microglía de las cortezas murinos neonatales por agitación mecánica con un agitador rotatorio. Este aislamiento microglia método rendimientos microglia corticales altamente puros que exhiben características morfológicas y funcionales indicativos de la microglia en reposo en condiciones normales, no patológicas in vivo. Este procedimiento también preserva el inmunofenotipo de la microglia y la funcionalidad bioquímica como se demuestra por la inducción de cambios morfológicos, la translocación nuclear de la subunidad p65 de NF-kappa B (p65), y la secreción de la citoquina proinflamatoria sello, factor de necrosis tumoral-α (TNF-α ), al lipopolisacárido (LPS) y Pam 3 CSK 4 (Pam) desafíos. Por lo tanto, el presente procedimiento de aislamiento conserva el inmunofenotipo de tanto reposo y Actimicroglia vada, proporcionando un método experimental de la investigación de la biología de la microglía en condiciones ex vivo.

Conservando el inmunofenotipo y la funcionalidad de la microglia ex vivo durante el aislamiento es crítico para ser capaz de utilizar estas células como un modelo de investigación para la biología de la microglia. Con el fin de demostrar la preservación con éxito de immunofunctionality microglia utilizando el presente método, se aislaron microglia corticales de los recién nacidos P3 (CX3CR1-GFP + / - y ratones C57BL / 6) y los cultivos tratados con LPS o Pam. Como...

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Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Una clave para el éxito de la investigación de la biología microglial es la preservación de immunofunction microglial ex vivo durante el aislamiento del tejido del SNC. Aislar microglia mediante agitación rotatoria resultados en cultivos de células de alta pureza y inmunofuncional según la evaluación de imágenes fluorescentes, inmunocitoquímica y ELISA después de la activación microglia con el lipopolisacárido estímulos proinflamatorias (LPS) y Pam 3 CSK 4 (Pam).

Aislamiento de cortical Microglia con Inmunofenotipo Conservas y funcionalidad de murinos Neonatos

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

isolation-cortical-microglia-with-preserved-immunophenotype

Research

JoVE Journal

Immunology and Infection

15.7K vistas.  Georgetown University Medical Center.  Una clave para el éxito de la investigación de la biología microglial es la preservación de immunofunction microglial ex vivo durante el aislamiento del tejido del SNC. Aislar microglia mediante agitación rotatoria resultados en cultivos de células de alta pureza y inmunofuncional según la evaluación de imágenes fluorescentes, inmunocitoquímica y ELISA después de la activación microglia con el lipopolisacárido estímulos proinflamatorias (LPS) y Pam...

Video: Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

El aislamiento de la microglía Primaria cultivos mixtos de células gliales del tejido neonatal cerebro de rata

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Investigación

Inmunología e Infección

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 ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia cultivo del Sistema Nervioso Central Neonatal y Adultos

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

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 ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Aislamiento de leucocitos a partir de los tejidos murinos en la interfase materno-fetal

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

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.

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 ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Análisis de linfocitos extravasación El uso de una<em&gt; In Vitro</em&gt; Modelo de la barrera sangre-cerebro humano

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

Aislamiento de cortical Microglia con Inmunofenotipo Conservas y funcionalidad de murinos Neonatos

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