Los autores desean agradecer a Anne Daron y Pierre Louis Loyher para asistencia editorial, la Plateforme Imagerie Pitié-Salpêtrière (PICPS) para obtener ayuda con el microscopio de dos fotones y el Fondo para el animal &quot;NAC&quot; y Camille Baudesson para los ratones que se reproducen asistencia. La investigación que lleva a estos resultados ha recibido financiación del Séptimo Programa Marco de la Comunidad Europea (FP7 / 2007-2013) en virtud del acuerdo de subvención n ° 304810 - RAID, y n ° 241440-Endostem, del Inserm, de la Université Pierre et Marie Curie &quot;Emergence &quot;, de la&quot; Ligue contre le cancer &quot;, de&quot; Asociación para la Investigación sobre el Cáncer le &quot;y de la&quot; Agence Nationale de la Recherche &quot;Aparición Programa 2012 (ANR-EMMA-050). PH recibió el apoyo de la &quot;Ligue contre le cancer&quot;.

NOTA: Todos los protocolos experimentales fueron aprobados por el Comité de Experimentación Animal y Ética francés y validados por el &quot;Servicio de Protección et Santé Animales, Environnement&quot; con el número A-75 hasta 2065. Los tamaños de muestra se eligen para asegurar la reproducibilidad de los experimentos, y de acuerdo con el 3R de la regulación ética animal. 1. Preparación del ratón <ol><li> Anestesia <ol><li> Para corto período de formación de imágenes (menos de 1 hr), anestesiar el ratón con una inyección intraperitoneal de 200 l de una solución salina que contiene ketamina (100 mg / kg) y xilazina (10 mg / kg). </li><li> Alternativamente, para el período de formación de imágenes más largo (hasta 4 horas), anestesiar el ratón con una inhalación de isoflurano 2,5% vaporizado en una mezcla 70/30 de O 2 / N 2 O, a través de una máscara adaptada. </li><li> NOTA: Este modo proporciona la inconsciencia más estable y evita la inyección intraperitoneal en serie de fármacos. </li&gt; <li> Una vez que el ratón está anestesiado, comprobar la inconsciencia a través de la estimulación de la pata con pinzas. </li></ol></li><li> Tinción Vascular / tinción adicional <ol><li> Para la tinción vascular: Inyectar por vía intravenosa en la vena de la cola, 200 l de rodamina-dextrano (2 MDA, 10 mg / ml en tampón de solución salina). </li><li> Para la tinción de neutrófilos: inyecte 2 g de Ly6G-PE (clon 1A8) en 100 l de solución salina por vía intravenosa en la vena de la cola. </li></ol></li><li> Inmovilización <ol><li> Para la inmovilización, utilizar un soporte estereotáctica por encargo adaptado al microscopio y al inhalador de gas anestésico. NOTA: El titular estereotáctica es un soporte de metal con dos soportes de placa de metal, que se está fijo y la otra extraíbles y adaptables, para apretar la cabeza del animal. </li><li> Instale la cabeza del ratón entre las placas de retención para asegurar el aislamiento de los movimientos respiratorios. Apriete los oídos entre placas de retención y la cabeza para estirar el sk. El registro para la estabilidad y la respiración bienestar del animal. </li></ol></li><li> Retire el cuero cabelludo <ol><li> Utilizar con cuidado etanol al 70% para mojar el cabello del cuero cabelludo con un aplicador estéril, evitando el contacto con los ojos. </li><li> Cortar la piel con las tijeras y alicates estériles para eliminar completamente el cuero cabelludo del retroceso del hueso parietal en el hueso frontal. Mantener alejado de hasta 3 mm de ojos y oídos para prevenir sangrado abundante. Retire el tejido conectivo laxo que cubre el cráneo (periostio). Lavar el cabello restante con una cálida toalla de papel empapada en PBS. </li></ol></li><li> Sistema de inmersión configurado <ol><li> Pegue un anillo de goma de 18 mm de diámetro con pegamento quirúrgico directamente en el cráneo usando una aguja de pequeño calibre para controlar la distribución. Pegamento de toda la periferia del anillo de goma para evitar fugas (30 l deberían ser suficientes). </li><li> Limpie el cráneo debajo del ring por última vez con una toalla de papel empapada en PBS y luego añadir 37 ° C PBS para la inmersión completa de lacráneo. Añadir una gota de NaCl 0.9% solución o ungüento oftálmico específico en cada ojo del ratón para evitar la sequedad de los ojos. Arrastre el titular estereotáctica con arreglo al objetivo. </li><li> Aproximadamente colocar el campo utilizando el ocular del microscopio a través de la transmisión directa. </li></ol></li></ol> 2. Dos fotones Imaging Acquisition <ol><li> Utilice un microscopio multifotónica junto con un láser de Ti: Cristal de zafiro, que proporciona 140fs pulsos de luz NIR, sintonizable selectivamente de 680 a 1080 nm, y un modulador acústico-óptico para el control de la potencia del láser. Utilice una configuración que incluye 3 detectores externos no descanned (NDD) (que permiten la grabación simultánea de 3 canales fluorescentes) con una combinación de 2 espejos dicroicos (565 nm y 690 nm), 565/610 y 500/550 filtros de paso de banda, y un filtro de paso corto 485, con un plan de apochromat × 20 (NA = 1) objetivo de inmersión en agua. </li><li> Establezca la cámara de calentamiento para permitir una buena homeotermia de los ANAESratón anestesiados., NOTA: La temperatura puede ajustarse de 1 hora antes de la formación de imágenes de sesión para una mejor estabilidad. En nuestro caso, 32 ° C ha sido elegido para obtener la temperatura corporal óptima del ratón anestesiado (36-37 ° C) y se mantiene hasta 4 horas. Esta temperatura puede ser adaptado para el sistema utilizado (sonda digital puede ser utilizado para comprobar este punto). Además, la cámara de calentamiento utilizado es oscuro / negro y cubre una parte del sistema (objetivos y la placa motorizada) para evitar la contaminación de luz externa. </li><li> Encienda el sistema <ol><li> Encienda el láser de Ti: Zafiro, entonces todo el sistema de microscopio. Inicie el software de adquisición. Ajuste el rango de longitud de onda del láser de 870 nm. Seleccione NDD apropiado para la grabación. NOTA: En nuestro caso, generación de segundo armónico (SHG) 19 y la proteína fluorescente cian (ECFP) son detectados por el DDN después del filtro de pase corto 485. ECFP también es detectado por el NDD después del filtro de paso de banda 500/550 y rodamina-dextrano es detectado por tél NDD después del filtro de paso de banda 565/610. </li></ol></li><li> Ajustar la configuración de adquisición <ol><li> Utilice el comando de imágenes &quot;adquisición en vivo&quot; del software y utilizar el control direccional microscopio, ajustar el enfoque en una región con ECFP y Rodamina señal. Ajustar la potencia del láser al mínimo para obtener la mejor relación señal a ruido con un mínimo de foto-daño. NOTA: El ajuste de la potencia que se utiliza varía según la profundidad de la zona de interés. Normalmente la configuración de OMA están distribuidas alrededor de un 20%, lo que representa una potencia de láser más allá del objetivo alrededor de 15 mW. Es mejor para aumentar el poder de ganancia en los PMT para cada NDD para reducir foto-blanqueo y foto-daño. Ajustes de adquisición típicos son la exploración individual con un tiempo de permanencia de píxel de 1,58 mS, y una resolución de 512 x 512 píxeles con un aumento de 1,5. </li></ol></li><li> Seleccione las regiones de interés <ol><li> Una vez que los parámetros de grabación se establecen, utilice de nuevo el comando de adquisición &quot;en vivo&quot;,y examinar el tejido para elegir un campo deseado (x, y, z) para monitorizar en tiempo real. NOTA: Por lo general, se optó por un campo que incluye tanto vascular y áreas del parénquima. </li><li> Registrar el campo con el comando de software &quot;posición&quot;. NOTA: Este comando memoriza x, y, z las coordenadas de los diferentes campos distantes. </li><li> Seleccionar y registrar campos de interés adicionales (hasta 3), utilizando el mismo procedimiento. </li><li> Establecer una pila Z 3D con el comando de software &quot;z-stack&quot; en la primera posición memorizada de acuerdo con el volumen requerido con aumento elegido. Memorizar posición más profunda primero, y luego la posición más alta. Normalizar la potencia del láser con la profundidad. NOTA: Para imágenes campo concomitante múltiple, adquirimos 5 rebanadas separadas por 3 micras z pasos para cada campo para adquirir un volumen de 12 micras de espesor. Estas configuraciones pueden ser adaptados para el tipo de célula estudiado. </li></ol></li><li> Adquisición de Lanzamiento <ol><li> Seleccione el220, el tiempo de la serie de comandos de software &quot;y seleccione la duración de intervalo de tiempo y el número de ciclos. Comience time-lapse de acuerdo con el lapso de tiempo y la duración requerida seleccionando el comando de software &quot;iniciar el experimento&quot;. NOTA: En nuestro caso, vamos adquiriendo un volumen cada 30 segundos durante 60 ciclos que representan a 30 min en tiempo real. Más pequeño lapso de tiempo se puede realizar para pista de rodadura celular rápido en el vaso sanguíneo. En este caso más delgado z-pila y no más de 2 campos diferentes se pueden grabar al mismo tiempo. </li><li> El control regular de la puesta en marcha. <ol><li> Siempre controlar al animal para asegurarse de que está inconsciente. NOTA: Sacudida de bigotes o las piernas del animal son indicadores de avivamiento. En el caso de la respiración del animal es carne seca, la cantidad de isoflurano se puede reducir ligeramente. La deriva de tejido fuerte durante la adquisición es un indicador del renacimiento del ratón. </li><li> Asegúrese de que el objetivo se sumerge correctamente. Renovar 37 ° C PBS between dos adquisiciones (30 min). NOTA: la desaparición progresiva de la señal durante la adquisición es indicador de PBS fugas. </li></ol></li></ol></li><li> Fin del procedimiento <ol><li> Una vez que se registran los videos requeridos, la eutanasia a los animales por dislocación cervical rápidamente antes de la reanimación. NOTA: la curación de heridas conduce a la formación de una costra en 24 a 48 horas lo que limita la posibilidad de realizar las imágenes longitudinal. </li></ol></li></ol> Análisis 3. Datos <ol><li> Corrección de la deriva <ol><li> Utilice software Imaris Bitplane para el seguimiento automático 3D. </li><li> Abra la secuencia de imágenes 3D en Imaris. Seleccione &quot;spot&quot; de comandos, y generar el seguimiento manual (clic del ratón + shift) para todos los puntos de tiempo por un mínimo de 4 puntos de anclaje diferentes, si es posible distribuida homogéneamente en el campo. </li><li> Aplicar la corrección de la deriva con el comando &quot;deriva correcta&quot; en la pestaña &quot;editar pista&quot;. </li><li> Compruebe tque la calidad de la corrección en x, y y z en particular para evitar la vacilación artefactual. NOTA: Si la corrección no es satisfactoria, pruebe otros puntos de anclaje o excluir el video del análisis. </li></ol></li><li> Seguimiento de la célula <ol><li> Utilice &quot;añadir nuevos puntos&quot; del sistema y seleccione &quot;puntos de la pista con el tiempo&quot; ajuste algoritmo. Vaya al paso siguiente. </li><li> Aplicar el procedimiento de seguimiento automático de células, ya sea en todo el canal de interés con el comando &quot;canal de origen&quot;. Objetos Set (es decir, las células) de diámetro a 10 micras. Vaya al paso siguiente. NOTA: La elección dependerá de la distinción entre los objetos y la especificidad de la señal medida. En resumen, el seguimiento automático de todo el canal es posible sólo si la señal detectada es específica para el objeto de interés. Debido a que tanto SHG y ECFP son detectados por el NDD después del filtro 485 pase corto, de seguimiento será más específica y eficiente utilizando la señal de ECFP registrada por el NDD después de la 500/550 filtros de paso de banda. Agrupados o objetos embalados densas requieren selección manual de objetos individuales similares al paso 3.1.2. </li><li> Seleccione el tipo de filtro &quot;de calidad&quot;, ajustar el umbral para excluir el seguimiento de objetos inespecíficos. Vaya al paso siguiente. </li><li> Elija &quot;browniano algoritmo Motion&quot; con parámetros precisos de &quot;distancia Max&quot; y &quot;tamaño Gap&quot; entre dos puntos. Validar generación pista. </li><li> Una vez que las pistas se calculan automáticamente, seleccione &quot;Duración pista&quot; tipo de filtro, ajustar el umbral para eliminar pistas más cortas que 120 segundos (lo que representa 4 puntos de tiempo). </li><li> Obligatorio: Después de seguimiento automático, comprobar la calidad de las pistas. <ol><li> Desenrolle la barra de tiempo para comprobar que los objetos siguen los caminos de pista. Si no, utilice las opciones de corrección de punto de seguimiento disponibles en la pestaña &quot;editar&quot;: Eliminar pistas falsas o incorrectas con &quot;borrar&quot; de comandos (borrar cualquier pista point individualmente con el comando &quot;desconexión&quot;). Si pista de atletismo es discontinua, añadir puntos de tiempo que faltan (clic con el ratón + shift), seleccione todos los puntos en una pista dada, y el uso de &quot;conectar&quot; de comandos en la pestaña &quot;editar pista&quot;. </li></ol></li></ol></li><li> Parámetros de cuantificación <ol><li> Seleccione la pestaña &quot;Estadísticas&quot; al mando &quot;Preferencias&quot;, seleccione los parámetros dinámicos de interés de la lista. Seleccione &quot;Exportar todas las estadísticas para presentar&quot; comando y guardar el archivo de Excel generado. NOTA: Varios parámetros dinámicos se obtienen directamente desde el software como longitudes de pista, velocidad instantánea, velocidad media, y un seguimiento de la rectitud. Coeficiente de Motilidad 6, 20 puede determinarse, pero no está directamente proporcionada por el software. </li><li> Para cada celda, calcular la media de los desplazamientos para cada período de tiempo (dt) (para una película de lapso de tiempo de 30 seg, dt será de 30, 60, 90 seg, etc).Trazar la media del desplazamiento cuadrado como una función del intervalo de tiempo. Obtener coeficiente de la motilidad mediante el cálculo de la pendiente de la parte lineal de la curva. </li></ol></li></ol>

<ol>
	<li>Ovchinnikov, D. A., 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=Expression+of+Gal4-dependent+transgenes+in+cells+of+the+mononuclear+phagocyte+system+labeled+with+enhanced+cyan+fluorescent+protein+using+Csf1r-Gal4VP16/UAS-ECFP+double-transgenic+mice.">Expression of Gal4-dependent transgenes in cells of the mononuclear phagocyte system labeled with enhanced cyan fluorescent protein using Csf1r-Gal4VP16/UAS-ECFP double-transgenic mice.</a> J Leukoc Biol. 83, 430-433 (2008).</li><li>Parihar, A., Eubank, T. D., Doseff, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytes+and+macrophages+regulate+immunity+through+dynamic+networks+of+survival+and+cell+death.">Monocytes and macrophages regulate immunity through dynamic networks of survival and cell death.</a> J Innate Immun. 2, 204-215 (2010).</li><li>Geissmann, F., Jung, S., Littman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+monocytes+consist+of+two+principal+subsets+with+distinct+migratory+properties.">Blood monocytes consist of two principal subsets with distinct migratory properties.</a> Immunity. 19, 71-82 (2003).</li><li>Robbins, C. S., Swirski, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multiple+roles+of+monocyte+subsets+in+steady+state+and+inflammation.">The multiple roles of monocyte subsets in steady state and inflammation.</a> Cell Mol Life Sci. 67, 2685-2693 (2010).</li><li>Shi, C., 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=Bone+marrow+mesenchymal+stem+and+progenitor+cells+induce+monocyte+emigration+in+response+to+circulating+toll-like+receptor+ligands.">Bone marrow mesenchymal stem and progenitor cells induce monocyte emigration in response to circulating toll-like receptor ligands.</a> Immunity. 34, 590-601 (2011).</li><li>Jacquelin, 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=CX3CR1+reduces+Ly6Chigh-monocyte+motility+within+and+release+from+the+bone+marrow+after+chemotherapy+in+mice.">CX3CR1 reduces Ly6Chigh-monocyte motility within and release from the bone marrow after chemotherapy in mice.</a> Blood. 122, 674-683 (2013).</li><li>Swirski, F. K., 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+of+splenic+reservoir+monocytes+and+their+deployment+to+inflammatory+sites.">Identification of splenic reservoir monocytes and their deployment to inflammatory sites.</a> Science. 325, 612-616 (2009).</li><li>Germain, R. N., Miller, M. J., Dustin, M. L., Nussenzweig, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+the+immune+system:+progress,+pitfalls+and+promise.">Dynamic imaging of the immune system: progress, pitfalls and promise.</a> Nat Rev Immunol. 6, 497-507 (2006).</li><li>Charo, I. F., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+roles+of+chemokines+and+chemokine+receptors+in+inflammation.">The many roles of chemokines and chemokine receptors in inflammation.</a> N Engl J Med. 354, 610-621 (2006).</li><li>Milo, I., 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=Dynamic+imaging+reveals+promiscuous+crosspresentation+of+blood-borne+antigens+to+naive+CD8++T+cells+in+the+bone+marrow.">Dynamic imaging reveals promiscuous crosspresentation of blood-borne antigens to naive CD8+ T cells in the bone marrow.</a> 122, 193-208 (2013).</li><li>Cavanagh, L. L., 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=Activation+of+bone+marrow-resident+memory+T+cells+by+circulating,+antigen-bearing+dendritic+cells.">Activation of bone marrow-resident memory T cells by circulating, antigen-bearing dendritic cells.</a> Nat Immunol. 6, 1029-1037 (2005).</li><li>Mazo, I. B., 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=Bone+marrow+is+a+major+reservoir+and+site+of+recruitment+for+central+memory+CD8++T+cells.">Bone marrow is a major reservoir and site of recruitment for central memory CD8+ T cells.</a> Immunity. 22, 259-270 (2005).</li><li>Travlos, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+structure,+function,+and+histology+of+the+bone+marrow.">Normal structure, function, and histology of the bone marrow.</a> Toxicol Pathol. 34, 548-565 (2006).</li><li>Mazo, I. B., 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=Hematopoietic+progenitor+cell+rolling+in+bone+marrow+microvessels:+parallel+contributions+by+endothelial+selectins+and+vascular+cell+adhesion+molecule+1.">Hematopoietic progenitor cell rolling in bone marrow microvessels: parallel contributions by endothelial selectins and vascular cell adhesion molecule 1.</a> J Exp Med. 188, 465-474 (1998).</li><li>Rashidi, N. M., 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=In+vivo+time-lapse+imaging+of+mouse+bone+marrow+reveals+differential+niche+engagement+by+quiescent+and+naturally+activated+hematopoietic+stem+cells.">In vivo time-lapse imaging of mouse bone marrow reveals differential niche engagement by quiescent and naturally activated hematopoietic stem cells.</a> Blood. , (2014).</li><li>Sipkins, D. A., 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=In+vivo+imaging+of+specialized+bone+marrow+endothelial+microdomains+for+tumour+engraftment.">In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment.</a> Nature. 435, 969-973 (2005).</li><li>Cariappa, A., 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=Perisinusoidal+B+cells+in+the+bone+marrow+participate+in+T-independent+responses+to+blood-borne+microbes.">Perisinusoidal B cells in the bone marrow participate in T-independent responses to blood-borne microbes.</a> Immunity. 23, 397-407 (2005).</li><li>Junt, 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=Dynamic+visualization+of+thrombopoiesis+within+bone+marrow.">Dynamic visualization of thrombopoiesis within bone marrow.</a> Science. 317, 1767-1770 (2007).</li><li>Zoumi, A., Yeh, A., Tromberg, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cells+and+extracellular+matrix+in+vivo+by+using+second-harmonic+generation+and+two-photon+excited+fluorescence.">Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence.</a> Proc Natl Acad Sci U S A. 99, 11014-11019 (2002).</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, 1869-1873 (2002).</li><li>Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choreography+of+cell+motility+and+interaction+dynamics+imaged+by+two-photon+microscopy+in+lymphoid+organs.">Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs.</a> Annu Rev Immunol. 26, 585-626 (2008).</li><li>Yost, C. C., 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=Impaired+neutrophil+extracellular+trap+(NET)+formation:+a+novel+innate+immune+deficiency+of+human+neonates.">Impaired neutrophil extracellular trap (NET) formation: a novel innate immune deficiency of human neonates.</a> Blood. 113, 6419-6427 (2009).</li><li>Devi, 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=Multiphoton+imaging+reveals+a+new+leukocyte+recruitment+paradigm+in+the+glomerulus.">Multiphoton imaging reveals a new leukocyte recruitment paradigm in the glomerulus.</a> Nat Med. 19, 107-112 (2013).</li></ol>

The authors have nothing to disclose.

Los puntos críticos de la metodología de formación de imágenes en vivo son para asegurar la estabilidad del foco a fin de maximizar la duración de la formación de imágenes y para reducir al mínimo el riesgo de contaminación bacteriana y la inflamación, lo que podría afectar la dinámica de las células inflamatorias. Imaging del hueso del cráneo ósea sigue estos objetivos como la cirugía realizada para obtener acceso a la médula ósea es mínima. El uso de material estéril y antisépticos...

The bone marrow plays a central role in hematopoiesis and represents the main reservoir of monocytes that constitutively recirculate between the blood and the medullar parenchyma, renew the pool of circulating monocytes with a short life span 2,3 and participate in the reconstitution of the steady state tissue-macrophages and dendritic cells 4. During inflammation or after transient aplasia, monocytes are actively mobilized from either the bone marrow or the spleen 5, 6, 7 and colonize inflamed organs. Several chemoattractant axis have been involved in the process of myeloid cell mobilization from the bone marrow 8, 5, 6,9. Beyond the myeloid compartment the bone marrow is also an important site of T lymphocyte priming 10 and a niche of immunological memory 11,12. Thus, this tissue is central for numerous investigations in the field of hematology and immunology. Our knowledge on the structural organization of medullar myeloid cells mainly arises from the analysis of histological section of fixed tissues 13. This static view does not allow for a study of the cellular exchange dynamic between the different compartments of the bone marrow, which is the basis of its functional activity.
Intravital imaging constitutes an important biological input in the study of cell mobility, cell adherence and cell-to-cell interactions, which were previously described only from in vitro systems. Technical challenges for proper intravital imaging include the ability to reach the tissue of interest in an optical point of view, and to maximize its isolation from physiological (breath, muscle or peristaltic contractions) or mechanical drifts (tissue disruption and extension following surgery, and exposure to microscope objective as well as temperature and vascular/oxygenation perturbations). Microscopic drifts may limit the ability to keep the focus a long time and could introduce artifacts in the quantification of cell motility. One alternative, validated for several tissues to reduce these technical difficulties, is to work on explanted tissue incubated in a thermostated and oxygenated medium; however, complete disruption of the lymphatic and vascular circulation may be problematic. Intravital imaging of skull bone marrow has several advantages concerning these issues. Firstly, it requires minimal surgical action. Secondly, thickness of the bone in this region allows direct visualization of bone marrow niches without abrasion, thus reducing physiological perturbations. The medullar network can be imaged in the parasagittal region of the bone; however the sinusoids are more visible in the fronto-parietal area where the bone matrix is thinner12,14. 
Intravital imaging relies on the availability of the most accurate fluorescent reporter tagging the population of interest. In vitro labelling of purified cell population before adoptive transfer led to important characterization of hematopoietic stem cell niches 15 or bone marrow endothelial microdomains favouring tumor engraftment 16, and provided several fundamental inputs on key concepts in immunology 17 . However, this approach usually requires hundreds of thousands of cells to get a chance to detect them afterwards in vivo. This could be explained by the high mortality rate following staining, the dilution in the whole body and the change in the activation state, which might lead to biased homing. Endogenous tagging from transgenic mouse system greatly overcomes these limitations and has allowed to image the behaviour of endogenous osteoblast 8, megakaryocytes 18 or myeloid-lineage subsets 6 . Nevertheless, one has to be cautious when considering the specificity of the fluorescent reporter among the studied subset. 
The Csf1r-Gal4VP16/UAS-ECFP, called MacBlue mouse 1, is a valuable transgenic system to study medullar monocytes with real time imaging 6. Intravenous injection of high molecular weight rhodamin-dextran distinguishes the medullar parenchyma from the vascular sinusoid network of the bone marrow. Using this approach, it is possible to track the monocyte behaviour in the different medullar compartments in a specific physiopathological context of interest. Furthermore, we propose an additional strategy to compare monocyte dynamics with that of neutrophils through in vivo labelling using a specific antibody.

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			<td>Surgical Glue, rubber ring fixation</td>
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tracking mouse bone marrow monocytes in vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

La estructura del cráneo del ratón ofrece una buena oportunidad para estudiar la fisiología de la médula ósea por intravital de imágenes. El hueso ser delgado alrededor de la zona frontal parietal, es posible obtener acceso a nichos medulares sin abrasión del hueso. La figura 1 representa un amplio campo 2D del cráneo de un ratón transgénico MacBlue. La matriz ósea está compuesta principalmente de colágeno I fácilmente detectable por SHG 19. La inyección de rodamina-dextrano ti...

Watch this Scientific Journal Video about Tracking Mouse Bone Marrow Monocytes In Vivo at JoVE.com

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Seguimiento de médula ósea de ratón monocitos<em&gt; En Vivo</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

tracking-mouse-bone-marrow-monocytes-in-vivo

Research

JoVE Journal

Immunology and Infection

Tracking Mouse Bone Marrow Monocytes In Vivo

9.4K vistas.  Sorbonne Universités, UPMC Univ Paris 06, CR7. Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism. 

Video: Tracking Mouse Bone Marrow Monocytes In Vivo

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Generación y Etiquetado de murino médula ósea procedentes de células dendríticas con nanocristales Qdot de Estudios de seguimiento

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Investigación

Inmunología e Infección

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

Diferenciar los roles funcionales de la expresión génica de las células inmunes y no inmunes en la colitis de ratón por Trasplante de Médula Ósea

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Aislamiento, Purificación y Etiquetado de neutrófilos de médula ósea de ratón de Estudios funcionales y experimentos de transferencia adoptivos

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Médula ósea derivada de macrófagos Producción

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Aislamiento y la inyección intravenosa de murino médula ósea derivados monocitos

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

Murino Hind Limb largo Bone La disección y el aislamiento de médula ósea

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Fémur Window Cámara Modelo de<em&gt; En Vivo</em&gt; Seguimiento de la célula de la médula ósea murina

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Clasificación de células activadas por fluorescencia para la purificación de células dendríticas plasmocitoide de la médula ósea de ratones

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Identificación y aislamiento de Oligopotent y linaje comprometido progenitores mieloides de la médula ósea de ratón

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

An &lt;em&gt;In Vivo&lt;/em&gt; Mouse Model to Measure Na&amp;#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Un modelo de ratón In Vivo a medida ingenuo CD4 células T activación, proliferación y diferenciación de Th1 inducida por las células dendríticas derivadas de la médula ósea

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...