Financiado por el programa intramuros de la Universidad de Servicios Uniformados.

1. La disección de los tejidos neonatal cerebro de rata <ol><li> 15 L-medios de comunicación acondicionado Chill Leibovitz (Leibowitz L-15 + BSA al 0,1% + 1% Pen / Strep) a 4 ° C. Medios calientes de cultivo (DMEM + 10% de SFB + 1% de penicilina / estreptomicina) a 37 ° C. Preparar 4 60x15 mm placas de Petri con 4-5 ml de los 15 L-acondicionado medios de comunicación sobre el hielo. Esterilice todos los instrumentos quirúrgicos con un 100% de etanol. </li><li> Enjuague los cachorros recién nacidos P2 rap con el 70% de etanol. (Crías de rata neonatal edades comprendidas entre P1-P5 se puede utilizar en este protocolo.) </li><li> Rápidamente decapitar a una cría de rata con tijeras estériles y colocar la cabeza de inmediato en etanol al 70%. Repetir para un total de 5 crías de rata luego transferir las cabezas en solución salina. </li><li> Retire los cerebros enteros de los jefes y colocarlos en una placa de Petri con 4 - 5 ml de solución de L-15 (Leibowitz L-15 + BSA al 0,1% + 1% Pen / Strep) en el hielo. </li><li> Repita los procedimientos de 1,2 - 1,4 para el resto de los cachorros en la camada. </li><li> Retire las meninges del cerebro y transferir el destejido cerebral IRED (es decir, la corteza, el cerebelo, el cerebro entero, etc) en una nueva placa de Petri con 4-5 ml de solución de L-15 (Leibowitz L-15 + BSA al 0,1% + 1% Pen / Strep) en el hielo. </li></ol> 2. Preparación de la población mixta de células gliales <ol><li> Sin dañando los tejidos cerebrales con unas tijeras o una cuchilla, la transferencia del tejido con una pipeta de 10 ml a un estéril de 50 ml tubo cónico. </li><li> Cónico de centrífuga a 2.500 RCF durante 5 min a 4 ° C. </li><li> Aspirar el sobrenadante. Añadir 4-5 ml de frescos 15 L-medios de comunicación (Leibowitz L-15 + BSA al 0,1% + 1% Pen / Strep). </li><li> Pipetear tejido hacia arriba y hacia abajo 10 veces con una pipeta estéril de 10 ml. </li><li> Colocar un filtro de células (100 poros micras) en un tubo cónico fresco 50 ml. Tejido pipeta arriba y hacia abajo una vez con una pipeta estéril de 5 ml y con la pipeta a ras de la filtro de células, dispensar el material a través del filtro de células en el tubo cónico. </li><li> Limpie el filtro de células con 4-5 ml de frescos 15 L-medios de comunicación (Leibowitz L-15 + 0,1% BSUna pluma + 1% / Strep). </li><li> Centrífuga cónico con células tensas a 2.500 RCF durante 5 min a 4 ° C. </li></ol> 3. Revestimiento y mantenimiento de cultivos mixtos de células gliales <ol><li> Para cada cerebro procesa las crías de rata, preparar 1 T-75 frascos estériles mediante la adición de 12 ml de medio de cultivo (DMEM + 10% de SFB + 1% de penicilina / estreptomicina) en cada frasco. </li><li> Aspirar el sobrenadante de las células sedimentadas y añadir 5-6 ml de medio de cultivo para el pellet celular. Pipetear arriba y hacia abajo 10 veces con una pipeta de 10 ml. </li><li> Transferir un volumen igual de suspensión de células en cada matraz T-75. </li><li> Incubar los frascos en un 5% de CO 2 incubadora a 37 ° C durante un total de 1-3 semanas. </li><li> Los matraces se incubaron primero durante 5 días, y el quinto día, reemplazar los medios de cultivo en cada matraz con 12 ml de medio fresco y volver a la incubadora. Luego, cada 3 días, vuelva a colocar los medios de comunicación acondicionado en cada frasco con 12 ml de medio fresco para lograr la confluencia. Esto se debe hacerne muy cuidadosamente sin tocar el fondo de los frascos donde las células se adhieren. </li></ol> 4. El aislamiento y forro de Primaria microglía <ol><li> Después de cultivos mixtos gliales son completamente confluente, eliminar matraces de la incubadora y cubrir las tapas matraz con parafilm para impedir el intercambio de gases con el aire ambiental. </li><li> Agitar frascos a 100 rps (compañero de laboratorio SI-600, Jeio Tech) durante 1 hora a 37 ° C. </li><li> Recoger los medios de comunicación de todos los frascos con una pipeta de 10 ml sin alterar la capa de los astrocitos en la superficie del frasco. Coloque el material en tubos de 50 ml cónicos. Añadir medio fresco a los frascos y frascos de retorno a la incubadora. </li><li> Centrifugar los tubos cónicos a 2.500 RCF durante 5 min a 4 ° C. </li><li> Aspirar el sobrenadante de todos los tubos cónicos. Gránulos de las células son células de la microglia alta pureza. Volver a suspender las pastillas en 1 ml de revestimiento microglia los medios de comunicación (DMEM + FES 10% + 1% de penicilina / estreptomicina). </li><li> Cuente con la densidad celular en los medios de comunicación que utilizan resuspendidasun hemocitómetro. </li><li> Añadir un volumen apropiado de los medios de comunicación de enchapado microglia para lograr un 2 x 10E5 células por ml de densidad. Placa como adecuado para el análisis experimental. </li><li> Permita que la microglia para unir toda la noche. </li></ol> 5. Los resultados representativos El protocolo descrito anteriormente resultados en cultivos primarios de alta pureza microglía. Según lo determinado por inmunohistoquímica para un marcador celular de macrófagos / microglia específico (Iba1), enchapados en culturas microglia son&gt; 90% de pureza (Figura 2). Además, la tinción de los marcadores de células de astrocitos, oligodendrocitos y neuronas específicas demuestra la mínima contaminación (Figura 2). Después de establecer cultivos mixtos de células gliales, la microglía puede ser aislado por agitación hasta 4 veces sucesivas. El primer aislamiento de microglia rendirá aproximadamente 2.2 x 106 células / ml o aproximadamente 9 millones de células por cada 10 frascos y el rendimiento esperado disminuye con cada sucesiva shake. Microglia aisló por este método se han utilizado con éxito para investigar la capacidad de fagocitosis, funciones tales como la producción de óxido nítrico, y la toxicidad neuronal (Figura 3) 13-14. <img alt="Figura 1" src="/files/ftp_upload/3814/3814fig1.jpg" /> Figura 1. Visión general del protocolo para la preparación de cultivos mixtos de células gliales y el aislamiento de la microglía primaria. <img alt="Figura 2" src="/files/ftp_upload/3814/3814fig2.jpg" /> Figura 2. El aislamiento de la microglía de alta pureza que se han verificado mediante análisis de inmunohistoquímica con la microglía (IBA-1, roja), astrocitos (GFAP, verde), las neuronas (NeuN, rojo) y oligodendrocitos (CC1, rojo) de marcadores específicos. La tinción de ADN de las células en cultivo por DAPI (azul) también se muestra. <img alt="Figura 3" src="/files/ftp_upload/3814/3814fig3.jpg" /> Figura 3. Microglia aislado en el método descricama se han utilizado en una serie de ensayos, incluyendo, pero no se limitan a, los ensayos de fagocitosis, ensayos de la función y los estudios de neurotoxicidad. En estos estudios, se ha encontrado que la microglía (IBA-1 positivo, verde), cuando se expone a controlar (A) o LPS (B) medios tratados, aumentar su fagocitosis de marcadas fluorescentemente perlas de látex (rojo), que pueden ser cuantificados ( C). Además, el LPS (1ng/ml) el tratamiento puede aumentar la producción de óxido nítrico en la microglía (D). Por último, tanto a través de transwell inserto separado o directa culturas microglia-neuronas, hemos encontrado que la microglia incubadas con LPS puede inducir la muerte celular neuronal, medido por la lactato deshidrogenasa (LDH) liberación (E, F).

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Neurochem. 76, 846-854 (2001).</li><li>Ni, M., Aschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+rat+primary+microglia:+isolation,+culturing,+and+selected+applications.">Neonatal rat primary microglia: isolation, culturing, and selected applications.</a> Curr. Protoc. Toxicol. Chapter 12, Unit 12.17 (2010).</li><li>Hassan, N. F., Rifat, S., Campbell, D. E., McCawley, L. J., Douglas, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+flow+cytometric+characterization+of+newborn+mouse+brain-derived+microglia+maintained+in+vitro.">Isolation and flow cytometric characterization of newborn mouse brain-derived microglia maintained in vitro.</a> J. Leukoc. Biol. 50, 86-92 (1991).</li><li>Hassan, N. F., Prakash, K., Chehimi, J., McCawley, L. J., Douglas, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+newborn+rabbit+brain-derived+microglia.">Isolation and characterization of newborn rabbit brain-derived microglia.</a> Clin. Immunol. Immunopathol. 59, 426-435 (1991).</li><li>Hassan, N. F., Campbell, D. E., Rifat, S., Douglas, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+human+fetal+brain-derived+microglia+in+in+vitro+culture.">Isolation and characterization of human fetal brain-derived microglia in in vitro culture.</a> Neuroscience. 41, 149-158 (1991).</li><li>Frank, M. G., Wieseler-Frank, J. L., Watkins, L. R., Maier, S. 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No hay conflictos de interés declarado.

Si bien este protocolo se utiliza rutinariamente para producir la microglía pura y saludable para los experimentos de investigación, la consideración cuidadosa de los aspectos técnicos durante las partes críticas del procedimiento minimizar la variabilidad en la microglia aislado. En primer lugar, durante la disección del tejido cerebral de crías de ratas neonatales, trabajando en el momento oportuno es necesario para minimizar el daño hipóxico isquémica, y en el tejido. Sin embargo, también es importante eli...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> 60 mm x 15 mm placas de Petri </td><td> Fisher de la marca </td><td> 0875713A </td><td></td></tr><tr><td> Afiladas tijeras de disección </td><td> Herramientas de Bellas Ciencia </td><td> 14094-11 </td><td></td></tr><tr><td> Dumont # 7b fórceps estándar consejos, curvas, 11cm </td><td> Herramientas de Bellas Ciencia </td><td> 11270-20 </td><td></td></tr><tr><td> Dumont # 5 fórceps estándar consejos, rectas, 11 cm </td><td> Herramientas de Bellas Ciencia </td><td> 11251-10 </td><td></td></tr><tr><td> 50 ml tubos de centrífuga cónicos </td><td> VWR </td><td> 89039-656 </td><td></td></tr><tr><td> 5 ml pipetas serológicas </td><td> Grenier Bio One </td><td> 606180 </td><td></td></tr><tr><td> 10 ml serológicas pipetas </td><td> Grenier Bio One </td><td> 607180 </td><td></td></tr><tr><td> 100 micras de nylon estéril filtro de células </td><td> Halcón </td><td> 35-2360 </td><td></td></tr><tr><td> 75 cm 2 frascos de cultivo de tejidos </td><td> Corning </td><td> 430641 </td><td></td></tr><tr><td> Medio esencial mínimo de Dulbecco (DMEM) </td><td> Gibco (Invitrogen) </td><td> 31053-028 </td><td></td></tr><tr><td> Leibovitz medio L-15 </td><td> Gibco (Invitrogen) </td><td> 11415064 </td><td></td></tr><tr><td> Suero bovino fetal </td><td> Gibco (Invitrogen) </td><td> 16000-036 </td><td></td></tr><tr><td> Fetal suero equino </td><td> Pescador </td><td> SH3007402 </td><td></td></tr><tr><td> La penicilina-estreptomicina </td><td> Gibco (Invitrogen) </td><td> 15140163 </td><td></td></tr><tr><td> 100% de etanol </td><td> La Compañía Warner Graham </td><td> 64-17-5 </td><td></td></tr><tr><td> Solución salina tamponada con fosfato, 10X, pH 7,4 </td><td> De calidad biológica, inc. </td><td> 119-069-131 </td><td> 1X en agua destilada estéril </td></tr><tr><td> Bolsas de bioseguridad </td><td> VWR </td><td> 14220-028 </td><td></td></tr><tr><td> Hemocitómetro </td><td> Hausser Científico </td><td> 1492 </td><td></td></tr><tr><td> 6-y las placas de cultivo celular con superficie Cellbind </td><td> Corning </td><td> 3335 </td><td></td></tr></tbody></table>

primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue

La microglía representan aproximadamente el 12% de la población celular total en el cerebro de los mamíferos. Mientras que las neuronas y astrocitos son considerados como los principales tipos de células del sistema nervioso, la microglía desempeñar un papel importante en la fisiología normal del cerebro mediante el control de los tejidos de los escombros y los agentes patógenos y el mantenimiento de la homeostasis en el parénquima a través de la actividad fagocítica 1,2. Microglia se activan durante una serie de condiciones lesiones y enfermedades, incluyendo enfermedad neurodegenerativa, lesión cerebral traumática, y la infección del sistema nervioso 3. Bajo estas condiciones de activación, la microglía aumentar su actividad fagocítica, sufren cambios morpohological y proliferativa, y secretan activamente las especies reactivas de oxígeno y nitrógeno, quimiocinas pro-inflamatorias y citoquinas, que a menudo la activación de un circuito paracrina o autocrina 4-6. A medida que estas respuestas microgliales contribuir a la patogénesis de la enfermedad en enfermedades neurológicas, la investigación se centró en microglia está garantizado. Debido a la heterogeneidad celular del cerebro, es técnicamente difícil obtener suficiente material muestra microglial con alta pureza durante los experimentos in vivo. La investigación actual sobre las funciones neuroprotectoras y neurotóxica de la microglía requieren un método de rutina técnica para generar constantemente microglia pura y saludable con el rendimiento suficiente para su estudio. Se presentan, en texto y video, un protocolo para aislar microglia primaria pura a partir de cultivos gliales mixtos para una variedad de aplicaciones posteriores. Brevemente, esta técnica utiliza tejido cerebral disociada de crías de rata neonatal para producir cultivos mixtos de células gliales. Después de que los cultivos gliales mixtas alcanzar la confluencia, microglía primarios son mecánicamente aislado del cultivo mediante un breve duración de agitación. La microglia se siembran con alta pureza para el estudio experimental. El principio y el protocolo de esta metodología han sidodescrito en la literatura 7,8. Además, las metodologías alternativas para aislar microglia primarias están bien descritos 9-12. Tejido cerebral homogeneizada puede ser separado por centrifugación en gradiente de densidad para producir primaria microglía 12. Sin embargo, la centrifugación es de longitud moderada (45 min) y puede causar daño celular y la activación, así como, causar enriquecido microglía y otras poblaciones celulares. Otro protocolo se ha utilizado para aislar microglía primaria en una variedad de organismos por prolongada (16 h) agitando mientras que en la cultura 9-11. Después de agitar, el sobrenadante se centrifuga medios para aislar microglía. Este método de aislamiento ya dos pasos también pueden perturbar la función microglial y la activación. Se utilizan principalmente el siguiente protocolo microglía aislamiento en nuestro laboratorio para un número de razones: (1) primaria microglía simular en la biología in vivo más fielmente que líneas celulares inmortalizadas roedores microglia, (2) Mecánico interrupción nominal minimiza disfunción potencial celular o activación, y (3) el rendimiento se puede obtener suficiente sin paso de los cultivos de células gliales mixtas. Es importante señalar que este protocolo utiliza el tejido cerebral de crías de rata neonatal para aislar microglia y que el uso de las ratas viejas para aislar microglía puede afectar significativamente el rendimiento, estado de activación, y las propiedades funcionales de aislado microglía. Hay evidencia de que el envejecimiento está relacionado con la disfunción microglia, neuroinflamación aumentado y patologías neurodegenerativas, estudios previos han utilizado para ex vivo para adultos microglia para comprender mejor el papel de la microglía en las enfermedades neurodegenerativas, donde el envejecimiento es un parámetro importante. Sin embargo, ex vivo microglía no se pueden mantener en cultivo durante períodos prolongados de tiempo. Por lo tanto, mientras que este protocolo se extiende la vida de la microglía primaria en cultivo, se debe señalar que la microglía comportan differently de los adultos microglia y en estudios in vitro deben ser cuidadosamente considerados cuando se traduce a un ajuste en vivo.

Watch this Scientific Journal Video about Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue at JoVE.com

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

Aislamiento de la microglía primaria a partir de la heterogeneidad celular del cerebro es esencial para investigar su papel en las dos condiciones fisiológicas y patológicas. Este protocolo describe un aislamiento mecánico y técnica mixta de cultivo celular que proporciona un alto rendimiento y alta pureza, viables células primarias microgliales para<em&gt; In vitro</em&gt; Estudio y aplicaciones posteriores.

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

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

primary-microglia-isolation-from-mixed-glial-cell-cultures-neonatal

Research

JoVE Journal

Immunology and Infection

39.1K vistas.  Uniformed Services University. Aislamiento de la microglía primaria a partir de la heterogeneidad celular del cerebro es esencial para investigar su papel en las dos condiciones fisiológicas y patológicas. Este protocolo describe un aislamiento mecánico y técnica mixta de cultivo celular que proporciona un alto rendimiento y alta pureza, viables células primarias microgliales para In vitro Estudio y aplicaciones posteriores.

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

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Un método simple y eficaz para aislar los macrófagos de la Mezcla de cultivos primarios de células de hígado en adultos

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Investigación

Inmunología e Infección

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

Generación de las Culturas Raft organotípicos de queratinocitos humanos primarios

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

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

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

Microglia cultivo del Sistema Nervioso Central Neonatal y Adultos

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

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

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

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

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

Isolation 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 ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

Aislamiento de células de Sertoli y peritubulares Las células de testículos de ratas

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Aislamiento y análisis de citometría de flujo de células inmunes del cerebro isquémico Ratón

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

La inducción de una respuesta inflamatoria en la Primaria hepatocitos culturas de ratones

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-&#954;B (NF-kB) and inhibitor &#954;B (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

Here, we present a protocol to isolate brain nuclei in the neonatal rat brain in conjunction with first colostrum feeding. This technique allows the study of nutrient insufficiency stress in the brain as modulated by enterocyte signaling.

Evaluación de estrés celular y la inflamación en discretos núcleos de cerebro secretoras de oxitocina en la rata Neonatal antes y después de la primera alimentación de calostro

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis.

The overall goal of this protocol is to instruct how to extract, maintain, and dissociate murine astrocyte and microglia cells from the central nervous system, followed by infection with protozoa parasites.

Aislamiento concomitante de astrocitos primarios y microglia para la infección por parásitos protozoos

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central ...