Este trabajo fue apoyado por la beca NS64571 del NINDS (CLH), por un premio de desarrollo de carrera a principios de la Clínica Mayo (CLH), y por un generoso regalo de Donald y Frances Herdrich (CLH). Nos gustaría agradecer a la Clínica Mayo de flujo básico Citometría de asistencia.

1. Inyección de virus intracraneal: La siguiente técnica ha sido modificada y utilizada ampliamente por nuestro laboratorio y sus colegas. En pocas palabras, la inyección intracraneana de la cepa de virus de Daniel murina de Theiler encefalomielitis (TMEV) o simulada-infección (1, 10) se lleva a cabo en ratones jóvenes (preferiblemente 5-6 semanas de edad) para obtener los leucocitos cerebro infiltrante (BILS). Tenga en cuenta que los resultados difieren entre la cepa de Daniel, la cepa Bean, y la cepa GDVII. Con el fin de la cosecha BILS, ratones reciben 2x10 5 PFU de TMEV y se infectan durante 24 horas. <ol><li> Coloque la aguja de inyección (calibre 27, ¼ en, Kendall), a una máquina de jeringa de 1 ml y se puso a Hamilton entregar 10 l de virus. </li><li> Elaborar TMEV en 10 l DMEM en la jeringa con especial atención a las burbujas de aire. Cuando sea apropiado, los ratones infectados farsa reciben 10 l de DMEM libre de virus. </li><li> Justo antes de la inyección, vierta aproximadamente 1 mL de isoflurano en una campana de cristal que contiene una malla de alambre con sustrato de algodón por debajo. </li><li> Ratones lugar direclty en la malla de alambre en la campana hasta que se convierten a la ligera anestesia, insensible a los pies-pinch e inmovilizado por el anestésico por inhalación, a unos 10-15 segundos. Los ratones no debe entrar en contacto directo con el isoflurano, la sustancia puede ser irritante y cáustico. </li><li> Mientras está bajo anestesia, para preparar a los ratones por inyección rápida localización de las coordenadas adecuadas en la región de la corteza frontal en el cráneo afeitado y con tinta. La ubicación general de inyección es de 1 mm anterior al bregma y 1 mm lateral a la sutura sagital en el lado derecho (Figura 1). Mientras que la inyección estereotáxica puede ser apropiada en algunas situaciones experimentales, hemos encontrado que a mano de inyección sin afeitar o tinta es adecuada para la mayoría de los experimentos. De separar el pelo con la punta de la aguja a lo largo de ambos ejes es apropiada para la inyección. </li><li> Para hacer una inyección, orientar la aguja perpendicular a la calavera y la prensa a través del hueso a una profundidad de aproximadamente 3 mm. </li><li> Expresar el virus en el cerebro al presionar el botón jeringa Hamilton abajo y esperar 2 segundos para que el virus para entrar en el cerebro antes de retirarse. </li><li> Retire con cuidado la aguja y coloque el ratón en una jaula limpia y seca. Los ratones despierta de la anestesia rápidamente y comiencen a funcionar normalmente en cuestión de minutos. Prestar la debida atención a los ratones que presentan síntomas anormales, tales como el sangrado excesivo en el sitio de inyección, como la eutanasia del animal puede ser apropiado. Animales adecuados de manejo 6 y el cumplimiento de los Institutos Nacionales de Salud y Cuidado de Animales institucional y las directrices del Comité de Uso deben ser seguidas. </li></ol> 2. Cerebro que infiltran la preparación de leucocitos: <ol><li> Prepare de fondo redondo de roble tubos de centrífuga de Ridge, que puede tener una capacidad de 30 ml con 10 ml de RPMI 1640, 9 de Percoll ml y 1 ml de PBS 10X y los tubos deben sentarse a temperatura ambiente en el banco durante el proceso de extracción del cerebro. </li><li> Traiga Ficoll-Paque Plus a temperatura ambiente (RT). </li><li> La eutanasia de animales modificados por sobredosis de isoflurano 5 y eliminar rápidamente el cerebro con una espátula de acero inoxidable y el tejido en un tubo cónico de 15 ml que contienen 5 ml de RPMI en el hielo. En algunas circunstancias, PBS-perfusión puede ser apropiado para eliminar las células circulantes en sangre periférica de la preparación BILS. Los ratones deben estar completa y correctamente anestesiado antes de la PBS-perfusión. </li><li> Transferencia de cerebros y la solución de RPMI a un molino de 7 ml de vidrio Pyrex tejido Tenbroeck marca y homogeneizar suavemente con 10-15 golpes. </li><li> Pipeta de un adicional de 5 ml de RPMI en el homogeneizador y pipeta de arriba y abajo dos veces. Transferencia de cerebros homogeneizado (aprox. 10 ml) para preparar con anterioridad de fondo redondo de tubo y suavemente invierta 2-3 veces para mezclar. </li><li> Giro homogenato de cerebro a 7800g ave durante 30 minutos a temperatura ambiente en un F0360 rotor de ángulo fijo en un Beckman Allegra X-22R centrífuga clínica. </li><li> Después de la centrifugación, eliminar los restos de mielina que ha flotado a la parte superior de la pendiente. Recoger la capa de leucocitos que flota por encima de los glóbulos rojos de pellets (Figura 2). </li><li> Cepa capa de leucocitos a través de un filtro de 40 micras de células en un Erlenmeyer de 50 ml. Llevar la solución a 50 ml con RMPI. </li><li> Tubos de centrifugado que contiene la suspensión celular diluida en una centrífuga clínica a 1500 rpm (600 g promedio) durante 5 min a temperatura ambiente. </li><li> Aspirar el sobrenadante y recoger el sedimento celular. Resuspender el precipitado en 1 ml de tampón FACS (1% de suero bovino azida de albúmina, 0,02% de sodio en el calcio y el magnesio libre de PBS) y la transferencia de la suspensión celular a 5 ml ronda final FACS tubos. </li><li> Con cuidado, subyace la suspensión con 1 ml de Ficoll-Paque Plus. </li><li> Girar el tubo a 2500 rpm (1400 dio) en centrífuga clínica durante 25 minutos a temperatura ambiente sin ningún freno. </li><li> Eliminar la capa blanca y esponjosa en la interfase (Figura 2) con una pipeta ml y traslado al nuevo tubo de 5 ml de FACS. Lávese las canas con 4 ml de tampón FACS. </li><li> Girar el tubo a 1500 rpm (600 g promedio) durante 5 min a temperatura ambiente en centrífuga clínica para precipitar las células. </li><li> Aspirar el sobrenadante y recoger el sedimento celular. Resuspender el pellet de leucocitos en un tampón adecuado y mantener en hielo en el nuevo tubo de FACS. </li><li> Recuento de células, evaluar la viabilidad celular por exclusión azul tripán y analizar por citometría de flujo. En general, los investigadores pueden esperar para adquirir aproximadamente 5 x 10 5 células / animal. </li></ol> 3. Inmunofenotipo por citometría de flujo <ol><li> Después de aislar y contar los leucocitos, las células del giro durante 3 minutos a 1500 rpm (600 dio) en una centrífuga clínica. </li><li> Resuspender el precipitado en tampón de bloqueo que contenga lo siguiente: tampón FACS, el sobrenadante del hibridoma 2.4G2 (Fc bloque; anti-CD16/32) y suero fetal bovino en una proporción de 10:05:01 y se incuba durante 30 minutos a 4 ° C . </li><li> Preparar y añadir anticuerpos conjugados contra los antígenos extracelular a las células bloqueadas en una concentración de 1:200 y se incuba durante 30 minutos a 4 ° C en la oscuridad. Mancha y se incuban las células en 200 l en un 96 y V-fondo de la placa. Controles apropiados incluyen células no teñidas y muestras de fluorocromo de compensación, según sea necesario. </li><li> Girar las células teñidas a temperatura ambiente durante 3 minutos a 1500 rpm (600 dio) en una centrífuga clínica utilizando un rotor adecuado para placas de 96 pozos. </li><li> Aspirar el sobrenadante y lavar las células con 200 l de tampón FACS pipeteando suavemente hacia arriba y abajo 3 veces. </li><li> Repita la técnica de lavado por encima de dos veces. </li><li> Después del lavado, resuspender las células en paraformaldehído al 2% y la transferencia a los tubos de FACS. La fijación es necesario para el análisis de citometría de flujo de las células infectadas de Bioseguridad Nivel 2 reactivos. </li><li> Ejecutar las células fijadas en un Calibur BD FACS siguiendo un método de citometría de flujo modificado 8 y analizar los archivos sin conexión utilizando FlowJo, WinMDI u otros programas disponibles y el análisis de citometría de flujo. </li><li> El análisis de 50-100,000 eventos por muestra se requiere generalmente para inmunofenotipo adecuada. </li></ol> Anticuerpos directamente conjugado utilizado en este experimento: CD45 se detectó con el clon 30-F11. Ly6C / G se detectó con el clon Gr1, RB6-8C5. CD11b fue detectado con el clon M1/70. F4/80 se detectó con el clon BM8. 4. Los resultados representativos: Mostramos los resultados de células de fenotipo BILS ratón a las 24 horas post-infección (Figura 3). Los leucocitos se tiñeron con PerCP-conjugadas anti-CD45 de ratón para detectar las células inmunes, PE-conjugado anti-ratón Ly6C / G para detectar monocitos inflamatorios, APC-conjugado anti-ratón CD11b y APC conjugado anti-ratón F4/80 para detectar células del linaje de los monocitos. El análisis se realizó con un instrumento BD FACS Calibur. <img src="/files/ftp_upload/2747/2747fig1.jpg" alt="Figura 1"/> Figura 1. Localización anatómica de la zona de inyección. El sitio se encuentra a 1 mm anterior al bregma y 1 mm lateral a la sutura sagital en el lado derecho. <img src="/files/ftp_upload/2747/2747fig2.jpg" alt="Figura 2"/> Figura 2. Los leucocitos ejemplo de separación de gradiente de leucocitos y BILS. Son inicialmente recogidos directamente por debajo de la capa de residuos de mielina y por encima de la pastilla de glóbulos rojos en el gradiente de Percoll. La capa blanca y esponjosa (BILS) en la interfaz se obtiene de la gradiente de Ficoll. <img src="/files/ftp_upload/2747/2747fig3.jpg" alt="Figura 3"/> Figura 3. Inmunofenotipo de cerebro infilatrating leucocitos a las 24 horas post-infección los leucocitos de ratón. Fueron aislados desde el cerebro por centrifugación diferencial de densidad de tejido homogeneizado preparado a partir de animales a las 24 horas después de la infección intracraneal. Las células se tiñeron con anticuerpos con fluorescencia conjugada contra CD45 de ratón, Ly6C / G, CD11b y F4/80. Gating inicial se realizó mediante el trazado de CD45, un marcador de las células inmunes, en contra de dispersión frontal (FSC), un indicador del tamaño de la celda (C). El hi CD45 (A, D), CD45 lo (B, E), y neg CD45 (F, G) la población se analizaron para determinar los niveles relativos de expresión de los marcadores de monocitos y macrófagos Ly6C / G, F4/80 (A, B , F), y CD11b (D, E, G). Ly6C / G + + CD11b + F4/80 monocitos inflamatorios se encuentra casi exclusivamente en la población CD45 alta (A, D), de conformidad con la infiltración de estas células de la periferia. Por el contrario, he aquí Ly6C/G-F4/80 CD11b macrófagos + se encuentra casi exclusivamente en la LO CD45 (B, E) y CD45 neg (G) las poblaciones, de acuerdo con un fenotipo residente. En numerosos experimentos, no hemos observado células CD45 de alta o Ly6C / G + + CD11b + F4/80 monocitos inflamatorios en el cerebro de los infectados o simulacro infecciónTed ratones (datos no mostrados).

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	<li>Buenz, E. J., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picornaviruses+and+cell+death.">Picornaviruses and cell death.</a> Trends Microbiol. 14, 28-36 (2006).</li><li>Buenz, E. J., Limberg, P. J., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+3-parameter+flow+cytometry-based+cell+death+assay.">A high-throughput 3-parameter flow cytometry-based cell death assay.</a> Cytometry A. 71, 170-173 (2007).</li><li>Deb, C., Lafrance-Corey, R. G., Zoecklein, L., Papke, L., Rodriguez, M., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demyelinated+axons+and+motor+function+are+protected+by+genetic+deletion+of+perforin+in+a+mouse+model+of+multiple+sclerosis.">Demyelinated axons and motor function are protected by genetic deletion of perforin in a mouse model of multiple sclerosis.</a> J. Neuropath. Exp. Neurol. 68, 1037-1048 (2009).</li><li>Deb, C., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKG2D+contributes+to+efficient+clearance+of+picornavirus+from+the+acutely+infected+murine+brain.">NKG2D contributes to efficient clearance of picornavirus from the acutely infected murine brain.</a> J. Neurovirol. 14, 261-266 (2008).</li><li>Donovan, J., &amp;amp, P. . B. r. o. w. n., , P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Euthanasia.">Euthanasia.</a> Current Protocols in Neuroscience. , A.4H.1-A.4H.4 (2005).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Handling+&#38;+restraint.">Handling &#38; restraint.</a> Current Protocols in Immunology. 73, 1.3.1-1.3.6 (2006).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parenteral+injections.">Parenteral injections.</a> Current Protocols in Neuroscience. , A.4F.1-A.4F.9 (2005).</li><li>Holmes, K. L., Otten, G., Yokoyama, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+analysis+using+Becton+Dickinson+FACS+Calibur.">Flow cytometry analysis using Becton Dickinson FACS Calibur.</a> Current Protocols in Immunology. , 5.4.1-5.4.22 (2002).</li><li>Howe, C. L., Ure, D., Adelson, J. D., LaFrance-Corey, R., Johnson, A., Rodriguez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+cells+directed+against+a+viral+peptide+contribute+to+loss+of+motor+function+by+disrupting+axonal+transport+in+a+viral+model+of+fulminant+demyelination.">CD8+ T cells directed against a viral peptide contribute to loss of motor function by disrupting axonal transport in a viral model of fulminant demyelination.</a> J Neuroimmunol. 188, 13-21 (2007).</li><li>Lipton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theiler's+virus+infection+in+mice:+An+unusual+biphasic+disease+process+leading+to+demyelination.">Theiler's virus infection in mice: An unusual biphasic disease process leading to demyelination.</a> Infect Immun. 11, 1147-1155 (1975).</li></ol>

Nosotros usamos la citometría de flujo para determinar la calidad de la preparación de la infiltración de las células cerebrales, y para distinguir las diferentes poblaciones de células inmunes 2, 9. En agudo puntos de tiempo, nuestro método BILS rendimientos altos porcentajes de monocitos inflamatoria dentro de la población CD45hi, así como un alto porcentaje de los macrófagos en la población CD45lo. Esto indica que una respuesta inmune reproducibles dentro del cerebro con firmeza se puede caracter...

<table border="1"><tbody><tr><th> Reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> TMEV </td><td> Howe laboratorio </td><td> N / A </td><td></td></tr><tr><td> Cepa de Daniel </td><td> Invitrogen </td><td> 21063-029 </td><td></td></tr><tr><td> isoflurano </td><td> Novaplus </td><td> NDC 0409-3292-49 </td><td></td></tr><tr><td> de fondo redondo de roble tubos de centrífuga de canto </td><td> Nalgene </td><td> 3118-0030 </td><td></td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 11875-093 </td><td></td></tr><tr><td> Percoll </td><td> GE Healthcare </td><td> 17-0891-02 </td><td></td></tr><tr><td> PBS 10 veces </td><td> Roche </td><td> 11666789001 </td><td></td></tr><tr><td> Ficoll-Paque Plus </td><td> GE Healthcare </td><td> 17-1440-02 </td><td></td></tr><tr><td> Azul de tripano al 0,4% (w / v) </td><td> Mediatech </td><td> 25 a 900-CI </td><td></td></tr><tr><td> 15 ml tubos cónicos </td><td> BD Falcon </td><td> 352097 </td><td></td></tr><tr><td> 7 ml de vidrio Pyrex marca Tenbroeck triturador de tejidos </td><td> Pescador </td><td> 08 a 414-10B </td><td></td></tr><tr><td> 40 m de células colador </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> Cónico de 50 ml </td><td> BD Falcon </td><td> 352070 </td><td></td></tr><tr><td> albúmina de suero bovino </td><td> Sigma </td><td> A9647 </td><td></td></tr><tr><td> azida de sodio </td><td> Sigma </td><td> S8032 </td><td></td></tr><tr><td> CMF-PBS </td><td> Mediatech </td><td> 21 a 040 CV </td><td></td></tr><tr><td> FACS tubos </td><td> BD Falcon </td><td> 352054 </td><td></td></tr><tr><td> de suero fetal bovino </td><td> Sigma </td><td> F4135 </td><td></td></tr><tr><td> 2.4G2 hibridoma </td><td> ATCC </td><td> HB-197 </td><td></td></tr><tr><td> Costar V-fondo de la placa </td><td> Corning </td><td> 3894 </td><td></td></tr><tr><td> Allegra X-22R centrífuga o equivalente </td><td> Beckman </td><td> N / A </td><td></td></tr><tr><td> De 96 pozos balde plato y rotor </td><td> Beckman </td><td> S2096 </td><td></td></tr><tr><td> Rotor de ángulo fijo </td><td> Beckman </td><td> F0360 </td><td></td></tr><tr><td> paraformaldehído </td><td> Sigma </td><td> P6148 </td><td></td></tr><tr><td> BD FACS Calibur </td><td> BD Biosciences </td><td> N / A </td><td></td></tr><tr><td> FlowJo 7.5 </td><td> Árbol Star, Inc. </td><td> N / A </td><td></td></tr><tr><td> CD45 </td><td> BD Biosciences </td><td> 557235 </td><td> clon: 30-F11 </td></tr><tr><td> Gr1 (Ly6C / G) </td><td> BD Biosciences </td><td> 553128 </td><td> clon: RB6-8C5 </td></tr><tr><td> CD11b </td><td> ebiosciences </td><td> 17-0112-83 </td><td> clon: M1/70 </td></tr><tr><td> F4/80 </td><td> ebiosciences </td><td> 17-4801-82 </td><td> clon: BM8 </td></tr></tbody></table>

isolation of brain-infiltrating leukocytes

Se describe un método para la preparación de los leucocitos cerebro infiltrante (BILS) de los ratones. Demostramos cómo infectar a los ratones con el virus de la encefalomielitis murina de Theiler (TMEV) a través de una técnica de inyección rápida intracraneal y la manera de purificar a la población de leucocitos enriquecido de infiltración de las células de todo el cerebro. En pocas palabras, los ratones son anestesiados con isoflurano en una cámara cerrada y son a mano alzada inyecta con una jeringa Hamilton en la corteza frontal. Los ratones son sometidas a muerte en varias ocasiones después de la infección por sobredosis de isoflurano y el cerebro entero se extraen y se homogeneizaron en RPMI con un triturador de tejidos Tenbroeck. Cerebro homogeneizado se centrifuga a través de un gradiente de Percoll continua del 30% para eliminar los restos celulares de mielina y otros. La suspensión celular Luego, se cuela a 40 micras, lavado y centrifugado en forma discontinua Ficoll-Paque Plus gradiente para seleccionar y purificar los leucocitos. Los leucocitos se lavan y se volvieron a suspender en los tampones apropiados para determinar el inmunofenotipo por citometría de flujo. La citometría de flujo revela una población de células del sistema inmune innato en las primeras etapas de la infección en ratones C57BL / 6. A las 24 horas post-infección, varios subconjuntos de células inmunes están presentes en el BILS, con una población enriquecida de Gr1 +, CD11b + y F4/80 + células. Por lo tanto, este método es útil en la caracterización de la respuesta inmunitaria a una infección aguda en el cerebro.

Watch this Scientific Journal Video about Isolation of Brain-infiltrating Leukocytes at JoVE.com

Isolation of Brain-infiltrating Leukocytes

Un método rápido para obtener la infiltración de leucocitos en el cerebro murino se describe. Este método utiliza un gradiente de Percoll continua y discontinua gradiente de Ficoll para seleccionar y purificar la capa de leucocitos enriquecido. Leucocitos aislados se puede caracterizar por las mediciones de citometría de flujo.

El aislamiento de la infiltración de leucocitos cerebro-

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

isolation-of-brain-infiltrating-leukocytes

Research

JoVE Journal

Immunology and Infection

19.0K vistas.  Mayo Clinic College of Medicine. Un método rápido para obtener la infiltración de leucocitos en el cerebro murino se describe. Este método utiliza un gradiente de Percoll continua y discontinua gradiente de Ficoll para seleccionar y purificar la capa de leucocitos enriquecido. Leucocitos aislados se puede caracterizar por las mediciones de citometría de flujo.

Video: Isolation of Brain-infiltrating Leukocytes

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

Aislamiento de células de ratón cavidad peritoneal

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Investigación

Inmunología e Infección

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Aislamiento de células inmunes funcionales cardíacos

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

Aislamiento y análisis de Brain-secuestrados Los leucocitos de<em&gt; Plasmodium berghei</em&gt; ANKA de ratones infectados

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Bioenergética y los Oxidativo ráfaga: Los protocolos para el aislamiento y la Evaluación de leucocitos humanos y plaquetas

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

El análisis de los efectos de las células del estroma en el reclutamiento de leucocitos de Flujo

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

Aislamiento de leucocitos de la interfaz humana Materno-fetal

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

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

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

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

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

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

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Aislamiento de leucocitos infiltrantes de ratón Piel Usando enzimática Digest y Separación de degradado

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

La recolección selectiva de leucocitos marginando-pulmonar

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

La recolección selectiva de leucocitos marginando-hepáticas

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...