Los autores desean agradecer al Dr. Alain Trautmann, que nos animó a realizar cortes de ganglios linfáticos. Este trabajo fue apoyado en parte por subvenciones de la Ligue Nationale Contre le Cancer, la Fondation pour la Recherche Médicale en Francia y la Asociación para la Investigación del Cáncer-sur-le.

1. Rebanadas de la preparación de los ganglios linfáticos <ol><li> Preparar un 4% de bajo punto de fusión solución de agarosa para incrustar. Diluir agarosa en PBS y microondas esta solución. Cuando la agarosa se disuelva por completo, mantener la solución a 37 ° C hasta que esté listo para su uso. </li><li> Prepare una placa de tejido de 6 pocillos. Añadir 1,1 ml de medio de cultivo RPMI completo por pozo, e insertar un filtro de organotípicos en cada pozo. Coloque la placa de 6 pocillos a 4 ° C hasta el momento de la transferencia de las rebanadas. </li><li> Coloque las arandelas de acero inoxidable, con un diámetro interior de 4 mm, en un plato de plástico llena de medio RPMI completo. Arandelas serán más utilizado para concentrar las células en el segmento de corte vibratome. </li><li> El sacrificio del ratón, de acuerdo con las normas locales de bienestar animal. Retire con cuidado los ganglios linfáticos periféricos del tejido circundante y colocarlas en un plato de plástico con helado de PBS. Se debe tener cuidado de no dañar la estructura de los ganglios linfáticos, ya que estos órganos son muy suaves. </li><li> Vierta el 4% en gel de agarosa en un plato de plástico de 35 mm y con delicadeza la transferencia de los nodos en el gel. Deje el gel de agarosa en hielo durante 5 min a endurecerse. </li><li> Retire el bloque de la antena y recortar el agar con el fin de salir de 3-5 mm de gel alrededor de cada uno de los ganglios linfáticos. </li><li> Conecte los nodos integrados en el disco de muestra del vibratome con adhesivo tisular no tóxico. Instale el disco muestra en la bandeja llena de helado de PBS. </li><li> La sección de agar de tejidos embebidos en el espesor de micras 320 con la velocidad vibratome conjunto en una serie lenta (0,3 mm / s) y la frecuencia de vibración establecidos en un rango medio (1,5 mm). </li><li> Con cuidado, la transferencia de las rebanadas de ganglios linfáticos, ya que se están reduciendo en los insertos de cultivo organotípico usando unas pinzas finas. Coloque 3 rebanadas en cada inserción. Tenga mucho cuidado en forma de filetes se pueden dañar fácilmente. Un nodo típico linfáticos periféricos producirán cinco secciones cuando se corta a 320 micras. Desechar las rodajas de primera y la última, ya que sólo contienen tejido superficial. </li><li> Coloque las arandelas de acero inoxidable en cada sector individual. Asegúrese de que las arandelas están bien posicionados en la agarosa alrededor del tejido. </li><li> Incubar la placa de cultivo a 37 ° C en un 5% de CO 2 humidificado incubadora hasta el momento de la placa de células T. </li></ol> 2. Aislar las células T de los ganglios linfáticos del ratón <ol><li> Aislar las células T de acuerdo con el artículo publicado anteriormente JoVe 3. </li><li> Utilizar las células T de inmediato y proceder a la siguiente sección. De lo contrario, los linfocitos pueden ser cultivadas durante la noche, en completo RPMI-medio suplementado con 10 ng / ml de IL-7. </li></ol> 3. Etiquetado de las células T aisladas <ol><li> Lavar las células en HBSS. Resuspender en el mismo tampón a 10 x 10 6 por ml. </li><li> Añadir el mismo volumen de HBSS conteniendo 1 mM celular Rastreador verde CMFDA dando una concentración final de 0,5 mM. </li><li> Incubar la suspensión de células a 37 ° C durante 5 min. Lavar las células dos veces con RPMI completo y medio plazo. </li><li> Resuspender las células a una concentración final de 10 x 10 6 por ml en RPMI completo y medio plazo. Estas células marcadas se sembraron en los cortes. </li></ol> Otros colorantes fluorescentes que CMFDA podría ser utilizado también, pero ten en cuenta que estas moléculas son potencialmente tóxicos por encima de ciertas concentraciones. 4. Revestimiento etiquetados células T en rodajas los ganglios linfáticos <ol><li> Retire el exceso de contenido medio en la parte interior de la lavadora con una pipeta. No permita que el tejido se seca, trabajar de forma rápida durante este paso. </li><li> Suavemente prescindir de 10 a 20 l etiquetados células T que corresponde a 1 a 2 x 10 5 células en la parte interior de la lavadora. Se debe tener cuidado de no tocar el tejido con la punta de la pipeta. Asegúrese de que la gota de suspensión celular se mantiene en su lugar. </li><li> Colocar las rebanadas con las células en la incubadora a 37 ° C y 5% de CO 2 por lo menos 30 minutos para permitir que los linfocitos migran hacia los tejidos. </li></ol> 5. Imágenes de las células T en rodajas los ganglios linfáticos En este video-artículo, las células T de imagen fluorescente en rodajas de ganglios linfáticos con un microscopio de campo amplio invertida y un microscopio confocal en posición vertical. <ol><li> Ajuste la temperatura a 37 ° C. Nuestros microscopios están equipados con cámaras de temperatura controlada. </li><li> Instalar un sistema de perfusión que permite la perfusión continua de la rebanada de ganglios linfáticos con oxigenada (5% CO2, 95% O 2) rojo fenol libre de medio RPMI. En nuestro sistema, los medios de comunicación perfusión entra en la cámara de imágenes de un lado por la gravedad. El medio es aspirado con una bomba conectada a un matraz de recogida de residuos. Establecer la velocidad de flujo de los medios de comunicación de 1 ml / min. </li><li> Con unas pinzas finas, delicadas sacar el trozo de la placa de 6 pocillos y se moje la preparación en caliente oxigenada medio RPMI durante unos segundos para deshacerse de células fluorescentes no se infiltraron en el tejido. </li></ol> Imaging células T con un microscopio invertido de gran campo <ol start="4"><li> Coloque el corte hacia abajo en una cámara de imágenes a medida que se compone de hilos de nylon pegado en la parte interior de un plato con fondo de cristal. En esta cámara, los hilos de nylon elevar el sector a partir de la copa y que la solución oxigenada de difundir en el tejido. Esto es necesario como la migración de las células T en los ganglios linfáticos es fuertemente dependiente de oxígeno. Asegurar el corte, añadiendo en él una arandela de acero inoxidable. En estas condiciones, el segmento se encuentra a unas pocas micras por encima del fondo del plato, lo que facilita la renovación de la solución de perfusión. </li><li> Para capturar un gran campo de visión (800 x 800 m, aproximadamente un tercio de la superficie de corte del nodo), recoger las imágenes con un lente de 10x objetivo seco. Se encontró que el 10 veces Nikon S fluor 0,5 NA era el más adecuado para nuestro análisis. </li></ol> Un típico experimento de time-lapse de imágenes ha capturado 5 aviones óptica que abarca una profundidad total de 50 micras en el axial (z) dimensión. Fluorescente las células T son generalmente imágenes en intervalos de entre 10 a 30 segundos durante 10 a 20 min. Imágenes de las células T con un microscopio confocal vertical El microscopio confocal utilizados en este protocolo es una Leica SP5 equipado con un objetivo de 20x de inmersión de agua (Olympus, 20x/0.95 NA). <ol start="6"><li> Asegurar la división para la etapa de formación de imágenes del microscopio. Nota: Por lo general, coloque una arandela en la preparación para inmovilizarlo. </li><li> Establecer una sesión de imágenes mediante la recopilación de una serie de imágenes a lo largo del eje z. A las células T de imagen en una estructura de tejido intacto, la posición de inicio se suele fijar entre 5 y 10 micras por debajo de la célula T primera etiqueta. </li></ol> 6. Los resultados representativos Siguiendo este video-protocolo que debe esperar para visualizar un gran número de células T fluorescentes acumuladas en la zona T del nodo, un fenómeno que ocurre normalmente en este entorno particular en vivo (Figura 1). En particular, las células T deben ser excluidos de las zonas de células B, que son por lo general sólo debajo de la cápsula. Un experimento exitoso también dará lugar a las células T reclutados en el tejido (Figura 2), mostrando un comportamiento muy móviles (Figura 3), en consonancia con los resultados publicados obtenidos en los ganglios linfáticos intactos 4. En promedio, la velocidad media de cada uno de las células T en lonchas deben ser cerca de 10 m / min (Figura 4). <img src="/files/ftp_upload/3054/3054fig1.jpg" alt="Figura 1"/> Figura 1. Las células T se acumulan en una rebanada de los ganglios linfáticos. Fluorescentemente marcado células T (CMFDA, verde) se han añadido a un ganglio linfático corte 30 minutos antes de la adquisición de imágenes (imagen superior). La imagen es la proyección máxima de 5 imágenes hasta los 50 m en la dirección z. La imagen de campo claro se muestra en la parte inferior. Las imágenes fueron capturadas con un microscopio de campo amplio. <img src="/files/ftp_upload/3054/3054fig2.jpg" alt="Figura 2"/> Figura 2. Las células T son reclutados en una porción de los ganglios linfáticos. Fluorescentemente marcado células T (CMFDA, verde) se han añadido a un ganglio linfático corte 30 minutos antes de la adquisición de imágenes con un microscopio confocal. Las imágenes fueron capturadas en la superficie de corte (foto superior) y 40 m por debajo (foto inferior). <img src="/files/ftp_upload/3054/3054fig3.jpg" alt="Figura 3"/> Figura 3. Las células T son muy móviles dentro de una porción de los ganglios linfáticos. Fluorescentemente marcado células T fueron estudiados durante 12 minutos usando un microscopio de campo amplio. Trayectorias individuales de las células T se muestran como un código de colores para representar las pistas desplazamientos cada vez mayor de azul (bajo número de células móviles) a rojo (células móviles de alta). Pistas se calcularon utilizando software Imaris. La línea blanca sigue el borde del nodo, mientras que el óvalo de puntos delimita la zona B de células putativo. <img src="/files/ftp_upload/3054/3054fig4.jpg" alt="Figura 4"/> Figura 4. Dentro de una porción de los ganglios linfáticos, la velocidad de las células T está cerca de 10 m / min. Las velocidades se calcularon utilizando software Imaris de pistas representado en la figura 3.

<ol>
	<li>Asperti-Boursin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCR7+ligands+control+basal+T+cell+motility+within+lymph+node+slices+in+a+phosphoinositide+3-kinase-independent+manner.">CCR7 ligands control basal T cell motility within lymph node slices in a phosphoinositide 3-kinase-independent manner.</a> J Exp Med. 204, 1167-1167 (2007).</li><li>Bhakta, N. R., Oh, D. Y., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+oscillations+regulate+thymocyte+motility+during+positive+selection+in+the+three-dimensional+thymic+environment.">Calcium oscillations regulate thymocyte motility during positive selection in the three-dimensional thymic environment.</a> Nat Immunol. 6, 143-143 (2005).</li><li>Matheu, M. P., Cahalan, M. D. <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+CD4++T+cells+from+mouse+lymph+nodes+using+Miltenyi+MACS+purification.">Isolation of CD4+ T cells from mouse lymph nodes using Miltenyi MACS purification.</a> J Vis Exp. , (2007).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1869 (2002).</li><li>Ehrlich, L. I., Oh, D. Y., Weissman, I. L., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+contribution+of+chemotaxis+and+substrate+restriction+to+segregation+of+immature+and+mature+thymocytes.">Differential contribution of chemotaxis and substrate restriction to segregation of immature and mature thymocytes.</a> Immunity. 31, 986-986 (2009).</li><li>Gollmer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL21+mediates+CD4++T-cell+costimulation+via+a+DOCK2/Rac-dependent+pathway.">CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway.</a> Blood. 114, 580-580 (2009).</li></ol>

Hemos descrito una técnica sencilla, rápida y robusta para la generación de cortes de ganglios linfáticos, que se utilizan para investigar el comportamiento de las células T introdujo. En los últimos años, este método ha sido aplicado con éxito el uso del timo y las rodajas de los ganglios linfáticos para identificar los factores extracelulares controlar el posicionamiento de las células T y la motilidad de 1,5. También se ha utilizado para medir las respuestas de Ca 2 + en timocitos durante la selección positiva y en las células T en el reconocimiento de antígenos 2,6. El ensayo parte de superposición presenta ventajas e inconvenientes que merecen ser discutidos. Es de destacar que este sistema permite el acceso a los tejidos, muy útil si se necesita para manipular farmacológicamente rebanadas con el fin de interferir con el control molecular de la migración celular. Con posterioridad a la imagen, las cortes pueden ser procesadas para inmunohistoquímica para recoger más información sobre las estructuras que han sido fotografiadas. Por otra parte, la observación se puede hacer con un microscopio de fluorescencia tradicionales de campo amplio. Aunque la resolución no es tan buena como con un microscopio confocal o de dos fotones, y que la fototoxicidad es, en principio, más grave, con un fotón que con el microscopio de dos fotones, tiene la ventaja de la simplicidad, un costo más bajo y mayor elección de longitudes de onda de excitación. La imagen de las células T en un ganglio linfático intacto requiere la inyección intravenosa de linfocitos marcados que, posteriormente, el hogar de los órganos linfoides. Como hemos demostrado anteriormente 1, el ensayo de corte es perfectamente compatible con los experimentos de transferencia adoptiva. Sin embargo, el tiempo necesario para que las células T a la casa de los ganglios linfáticos pueden complicar el uso de colorantes fluorescentes que tienen una tendencia a salirse de las células a través del tiempo. Este es el caso de la Ca 2 + colorante fura-2. Con el reclutamiento rápido (&lt;30 min) de las células T en el tejido, el ensayo de corte ofrece la oportunidad de usar estos colorantes. Finalmente, este método tiene la gran ventaja de analizar las funciones de las células T en varios tejidos humanos mantiene viva. Este sistema experimental también presenta limitaciones que deben tenerse en cuenta. Los daños asociados con el corte pueden afectar el funcionamiento de las células T, especialmente en la región superficial del tejido cerca de la superficie de corte. A fin de evaluar la muerte celular en el sector, hemos utilizado el colorante fluorescente verde SYTOX que penetra en las células con la membrana plasmática comprometida. Nuestros experimentos muestran que alrededor del 20% del total de las células ganglionares, en su mayoría localizadas en la región superficial del tejido, fueron etiquetados con fluorescencia con este colorante nuclear. Otro problema potencial con el ensayo es la capacidad de los sectores para conservar importantes factores solubles como las quimiocinas. Aunque, no tenemos ninguna indicación de que nuestros datos se vieron afectados por este tipo de problemas ya que las células T muestran una buena movilidad en el sector, nos gustaría hacer hincapié en la importancia de la imagen de las células T en las regiones de salud ubicados en varias decenas de micrones de la superficie de corte. Considerando que, con esto se podría hacer con los microscopios tradicionales (o de campo amplio confocal), como en este protocolo, es probable que el ganglio linfático rebanada preparación combinada con dos fotones de imagen aumentará la resolución espacial en profundidad y reducir la fototoxicidad.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> Microscopio confocal </td><td> Leica </td><td> SP5 </td><td></td></tr><tr><td> Vibratome </td><td> Leica </td><td> VT1200S </td><td></td></tr><tr><td> Pinzas finas </td><td> Mundial de Instrumentos de Precisión </td><td> 14142 </td><td></td></tr><tr><td> 30 inserciones de Cultura mm </td><td> Millipore </td><td> PICM0RG50 </td><td></td></tr><tr><td> RPMI </td><td> Invitrogen </td><td> 61870010 </td><td> Completo RPMI-medio se realiza mediante la adición de un 10% inactivado por calor suero fetal bovino y penicilina / estreptomicina </td></tr><tr><td> Solución salina equilibrada de Hanks (HBSS) </td><td> Invitrogen </td><td> 14170088 </td><td></td></tr><tr><td> Baja la temperatura de gelificación de agarosa, el tipo VII-A </td><td> Sigma-Aldrich </td><td> A0701 </td><td></td></tr><tr><td> Cianoacrilato de butilo pegamento, Vetbond </td><td> 3M </td><td> 1469 </td><td></td></tr><tr><td> Arandelas de acero inoxidable, 4 mm de diámetro interior </td><td> Bricolaje </td><td></td><td></td></tr><tr><td> Rastreador de células verdes CMFDA </td><td> Invitrogen </td><td> C7025 </td><td></td></tr></tbody></table>

ex vivo imaging of t cells in murine lymph node slices with widefield and confocal microscopes

Células T naive continuamente el tráfico de órganos linfoides secundarios, incluyendo los ganglios linfáticos periféricos, para detectar antígenos expresados ​​raro. La migración de las células T en los ganglios linfáticos es un proceso complejo que implica factores celulares y químicos, incluyendo las quimioquinas. Recientemente, el uso de la microscopia de dos fotones ha permitido hacer un seguimiento células T en los ganglios linfáticos intactos y para obtener alguna información cuantitativa sobre su comportamiento y sus interacciones con otras células. Mientras que hay ventajas obvias para un sistema in vivo, este enfoque requiere una instrumentación compleja y costosa y ofrece un acceso limitado a los tejidos. Para analizar el comportamiento de las células T en los ganglios linfáticos murino, hemos desarrollado un ensayo de corte 1, originalmente creado por los neurobiólogos y adaptado recientemente a murino timo 2. En esta técnica, marcado con fluorescencia células T se colocan en la parte superior de una porción de ganglios linfáticos extremadamente preparado. En este video-artículo, la localización y la migración de las células T en el tejido se analiza en tiempo real con un campo amplio y un microscopio confocal. La técnica que complementa en vivo de dos fotones microscopía ofrece un método eficaz para la imagen las células T en su entorno natural y para dilucidar los mecanismos subyacentes de migración de las células T.

Watch this Scientific Journal Video about Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes at JoVE.com

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Este protocolo describe un método para células T de imagen fluorescente introducido en rodajas los ganglios linfáticos. La técnica permite en tiempo real, análisis de la migración de las células T con las tradicionales de fluorescencia de campo amplio o microscopios confocal.

Ex vivo de imágenes de células T en los ganglios linfáticos murino Rebanadas con campo amplio y microscopios confocal

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

ex-vivo-imaging-t-cells-murine-lymph-node-slices-with-widefield

Research

JoVE Journal

Immunology and Infection

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

16.7K vistas.  Université Paris Descartes, CNRS (UMR 8104). Este protocolo describe un método para células T de imagen fluorescente introducido en rodajas los ganglios linfáticos. La técnica permite en tiempo real, análisis de la migración de las células T con las tradicionales de fluorescencia de campo amplio o microscopios confocal.

Video: Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Transducción retroviral de receptores de células T en ratones las células T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Investigación

Inmunología e Infección

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Microscopia intravital de los ganglios linfáticos inguinales

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Murine Superficial Lymph Node Surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

Murino linfáticos superficiales Cirugía de los ganglios

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Imagen intravital del nodo linfático poplíteo ratón

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

Generación de nódulos linfáticos de grasa Pad quimeras para el Estudio del Ganglio Linfático estroma origen de célula

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

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 ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Una<em&gt; Ex vivo</em&gt; Modelo de un ataque de células T Oligodendrocyte dirigida en Rebanadas cerebral agudo

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

Ex Vivo Infección de murino Epidermis con virus herpes simplex tipo 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

Aislamiento y<em&gt; Ex Vivo</em&gt; Cultura de Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; Las células γδ T, una célula αβT Progenitor Extrathymic

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

Murina de linfocitos Etiquetado por<sup&gt; 64</sup&gt; Cu-Anticuerpo objetivo de receptores de<em&gt; En Vivo</em&gt; Tráfico de células mediante PET / CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...