Este trabajo fue financiado por una beca de investigación y una beca en la ayuda del Proyecto de Nanotecnología de Alimentos del Ministerio de Agricultura, Silvicultura y Pesca de Japón.

1. La perfusión del hígado <ol><li> Anestesiar a un macho adulto de rata Sprague-Dawley (de 10 a 15 semanas de edad, peso corporal 150-200 g) mediante una inyección intraperitoneal de una solución de pentobarbital sódico (50 mg de sodio pentobarbital / kg de peso corporal). </li><li> Colocar el animal en un recipiente de acero y gas etanol al 70% en el abdomen y el tórax. </li><li> Hacer un pequeño corte y retire la piel. Abrir el abdomen por el corte con una tijera. </li><li> Confirmar la ubicación de la vena porta, pasar un hilo quirúrgico en la vena porta y se agrupan libremente. </li><li> Grupo la parte distal de la vena cava inferior y la vena mesentérica para prevenir el reflujo de sangre hacia el hígado. Abrir la cavidad torácica por el diafragma de corte con las tijeras, y exponer el corazón. </li><li> Insertar un catéter de plástico (2 mm de diámetro) en la vena porta y apretar firmemente el hilo alrededor del catéter. </li><li> Conectar el catéter a un sistema de perfusión que se carga con solución de lavado (Ca 2 +-Mg 2 + libre HBSS que contenía 0,5 mM EGTA, 10 mM HEPES y 4,2 mM NaHCO 3, pH 7.2) previamente calentada a 37 ° C. </li><li> Comenzar la perfusión in situ a una velocidad de 10 ml / min. Al mismo tiempo, hacer un corte en la pared de la aurícula derecha. Continuar la perfusión durante 10 minutos. </li><li> Cambie la solución de perfusión a la solución de colagenasa [HBSS que contiene HEPES 10 mM y 4,2 mm de NaHCO3 complementados con colagenasa tipo I (0,05%) y los inhibidores de tripsina (50 ug / ml), pH 7.5], y la perfusión de 10 a 20 minutos a una velocidad de 10 ml / min hasta que el hígado se inflama y se muestra un poco el signo de la desintegración de la estructura del hígado debajo de la membrana serosa. </li></ol> 2. Preparación de la fracción del parénquima celular de hepatocitos ricos <ol><li> Retire con cuidado la transferencia del hígado en un vaso estéril que contiene 25 ml de solución de colagenasa. Picar en trozos pequeños con tijeras. </li><li> Añadir 75 ml de MEM frío en la suspensión celular resultante. Después de pipeteo suave, filtrado a través de un colador celular (100 m) para eliminar los tejidos conectivos y los fragmentos de tejido sin digerir. </li><li> Transferir el filtrado en tubos de 50 ml y se centrifuga a 50 g durante 1 min a 4 ° C (freno desactivado). </li><li> Elimine con cuidado el sobrenadante. Resuspender el precipitado con el MEM frío. </li><li> Repita 2,4) tres veces. </li></ol> 3. Cultura mixta primaria de las células hepáticas <ol><li> Suspender las células obtenidas en 2.5) en el medio de cultivo compuesto por DMEM con 10% de FCS inactivado por calor, suplementado con 100 mM β-mercaptoetanol, 10 mg / ml de insulina, 100 ug / ml de estreptomicina y 100 U de penicilina / ml, y la semilla en nueve y cincuenta y cinco frascos de cultivo de tejido (con una superficie: 75 cm 2) a una densidad de 6,7 x 10 4 células / cm 2 (5 x 10 6 células / frasco / 12 ml -15 de media). </li><li> Incubar la cultura flasksat 37 ° C en una atmósfera de 5% de CO 2 a 95% de aire. </li><li> Vuelva a colocar el medio de cultivo cada 2 ó 3 intervalos de días. </li></ol> 4. Aislamiento selectivo de los macrófagos <ol><li> Después de 12 días de cultivo, los macrófagos proliferan en la capa celular. Estas células son suspendidas por mutuo temblor de los frascos de cultivo de 80 a 120 golpes por minuto durante 30 minutos a 37 ° C. </li><li> Transferencia al medio de cultivo en 100 mm no de cultivo de tejido de plástico platos grado. Piscina del medio a partir de dos frascos T75 en un plato de plástico. Frascos de rellenar la cultura con el medio de cultivo y devolverlos a la incubadora. </li><li> Incubar los platos de plástico por 30 minutos a 37 ° C en una atmósfera de 5% de CO 2 a 95% de aire. Macrófagos fácilmente se adhieren a los platos de cultivo de tejidos no de plástico de grado en este proceso de incubación, mientras que otras células contaminantes no. </li><li> Aspirar el medio y enjuague suavemente el plato de plástico con PBS. Repita este paso tres veces. </li><li> Añadir 1 ml de solución TrypLE Express en el plato, y se incuba durante 10 minutos a 37 ° C en una atmósfera de 5% de CO 2 a 95% de aire. </li><li> Añadir 9 ml del medio de cultivo. Raspe suavemente los macrófagos unida por un rascador de células. </li><li> La transferencia de la suspensión de células en un tubo cónico de 15 ml y centrifugar a 180 xg durante 5 min a 25 ° C (freno). </li><li> Eliminar el sobrenadante y añadir 1 ml de medio de cultivo. Se disocian en células individuales mediante pipeteo vigoroso. Enumerar el número de células por un hemocitómetro de conteo. </li><li> Medidas de aislamiento se puede repetir con los frascos de cultivo mismo de dos a intervalos de tres días por más de dos semanas. </li><li> Macrófagos aislados pueden ser cultivados en el medio de cultivo se describen en 3.1. </li></ol> 5. Resultados representante Un ejemplo de una cultura mixta primaria de adultos células de hígado de rata se muestra en la Figura 2. Hepatocitos del parénquima perder la morfología de las células epiteliales dentro de unos días en la cultura, degenerada o transformarse en células similares a fibroblastos.Luego, alrededor del día 6 al 12 de contraste de fase brillante, redondo como los macrófagos-células vigourouly proliferan en la lámina de células de fibroblasto. Por agitación de los frascos de cultivo, los macrófagos son fácilmente suspendidas en el medio de cultivo, y la posterior transferencia e incubación en platos de plástico resultar en una adhesión selectiva de los macrófagos (Figura 3). Las células como los macrófagos-se puede cosechar a partir del día 8, y el número llegó a niveles máximos en los días de 12 a 14. Más de 10 6 células se pueden cosechar en un frasco de cultivo T75 repetidamente de 2 a 3 días a intervalos de más de dos semanas, lo que permite un rendimiento total de células por frasco de frasco de cultivo de 10 7 por T75 (Figura 3). La pureza de los macrófagos aislados de 95 a 99%, según lo evaluado por citometría de flujo o inmunocitoquímica con ratas macrófagos anticuerpos específicos, tales como ED-1 (Figura 4), ​​ED-3, OX-41 (Figura 4) y 9 Iba1. Las células aisladas muestran características típicas de los macrófagos, como la fagocitosis activa de cuentas polystylene (Figura 5), la respuesta proliferativa a GM-CSF recombinante, la secreción de antiinflamatorios / anti-citoquinas inflamatorias a la estimulación con LPS, y la formación de células gigantes multinucleadas 9. <img src="/files/ftp_upload/2757/2757fig1.jpg" alt="Figura 1" /> Figura 1. Un esquema para el aislamiento selectivo y la purificación de los macrófagos-células de cultivo mixto primarios de células de hígado de rata. <img src="/files/ftp_upload/2757/2757fig2.jpg" alt="Figura 2" /> Figura 2. Cultivo primario de células de hígado de rata y la proliferación de los macrófagos-células. La flecha indica una célula gigante multinuclear. Barra de escala = 100 micras. Reimpresión de la Revista de Métodos inmunológicos, Vol.360, 47-55 (2010) 9, con permiso de Elsevier. <img src="/files/ftp_upload/2757/2757fig3.jpg" alt="Figura 3" /> Figura 3. Aislamiento selectivo de los macrófagos-células de la agitación y el método de fijación. Las células fueron suspendidas en el medio de cultivo agitando los frascos, posteriormente trasladados en cajas de no cultivo de tejidos de plástico de grado, y se incuba a 37 ° C. A los 10 min después de la siembra, como los macrófagos-células pegadas a la superficie del plato, mientras que otras contaminación de las células fibroblásticas quedó suspendida. Después de lavar con PBS, una población de macrófagos altamente purificada se obtuvo. Estas células ganó la morfología típica de los macrófagos después de 40 min de la cultura, y las células mitóticas se observaron con frecuencia (flechas). Los cambios posteriores en el número de células recuperadas de los frascos en los períodos de cultura diferente se muestran. Los valores son un promedio ± SD desde tres hasta cinco frascos. Los experimentos se repitieron tres veces y los datos se indican con símbolos diferentes. Barra de escala = 100 micras. Reimpresión de la Revista de Métodos inmunológicos, Vol.360, 47-55 (2010) 9, con permiso de Elsevier. <img src="/files/ftp_upload/2757/2757fig4.jpg" alt="Figura 4" /> Figura 4. Coloración inmunocitoquímica de los macrófagos-células con anticuerpos monoclonales contra los macrófagos de rata. <img src="/files/ftp_upload/2757/2757fig5.jpg" alt="Figura 5" /> Figura 5. Fagocitosis de FITC microesferas por los macrófagos-células.

<ol>
	<li>Crispe, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+liver+as+a+lymphoid+organ.">The liver as a lymphoid organ.</a> Annu. Rev. Immunol. 27, 147-163 (2009).</li><li>Roberts, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+Kupffer+cell+in+mediating+hepatic+toxicity+and+carcinogenesis.">Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis.</a> Toxicol. Sci. 96, 2-15 (2007).</li><li>Gao, B., Jeong, W. I., Tian, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver:+An+organ+with+predominant+innate+immunity.">Liver: An organ with predominant innate immunity.</a> Hepatology. 47, 729-736 (2008).</li><li>Janousek, J., Strmen, E., Gervais, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+murine+Kupffer+cells+by+centrifugal+elutriation.">Purification of murine Kupffer cells by centrifugal elutriation.</a> J. Immunol. Methods. 164, 109-117 (1993).</li><li>Olynyk, J. K., Clarke, S. L. <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+primary+culture+of+rat+Kupffer+cells.">Isolation and primary culture of rat Kupffer cells.</a> J. Gastroenterol. Hepatol. 13, 842-845 (1998).</li><li>Heuff, G., Meyer, S., Beelen, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+rat+and+human+Kupffer+cells+by+a+modified+enzymatic+assay.">Isolation of rat and human Kupffer cells by a modified enzymatic assay.</a> J. Immunol. Methods. 174, 61-65 (1994).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Methods Cell Biol. 13, 29-83 (1976).</li><li>Yoshioka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+expression+of+cytokine+mRNAs+by+lipopolysaccharide+in+bovine+Kupffer+cells.">Primary culture and expression of cytokine mRNAs by lipopolysaccharide in bovine Kupffer cells.</a> Vet. Immunol. Immunopathol. 58, 155-163 (1997).</li><li>Kitani, H., Takenouchi, T., Sato, M., Yoshioka, M., Yamanaka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+isolation+method+for+macrophage-like+cells+from+mixed+primary+cultures+of+adult+rat+liver+cells.">A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells.</a> J. Immunol. Methods. 360, 47-55 (2010).</li><li>Gorrell, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+fibrosis:+the+hepatocyte+revisited.">Liver fibrosis: the hepatocyte revisited.</a> Hepatology. 46, 1659-1661 (2007).</li></ol>

Todos los animales fueron mantenidos y operados en las instalaciones de los animales en el Instituto Nacional de Salud Animal, de acuerdo con las directrices y regulaciones establecidas por el Comité de la Institución para los experimentos con animales.

Aquí, se presenta un método simple y eficiente para obtener los macrófagos de la mezcla de culturas primarias de adultos células de hígado de rata. Nuestro método se basa primero en la novedosa actividad proliferativa de macrófagos en el cultivo mixto de células de hígado de rata, y posterior aislamiento y purificación de estas células sobre la base de sus características biológicas como los macrófagos 9. Puede ser posible que los macrófagos proliferan en el cultivo mixto primarios de células de hígado en adultos podría haberse originado a partir de Kupffer o las células relacionadas que contaminó en la fracción del parénquima hepatocitos. Los macrófagos podrían haber respondido a determinados cambios ambientales provocados por la transformación de las células del parénquima hepatocitos fibroblástica 10 y otros en los cultivos primarios mixtos. En conclusión, el método proporciona un aislamiento de los macrófagos-células en cantidad suficiente y la pureza del cultivo primario de hígado de rata cellswithout utilizando sofisticados equipos y conocimientos técnicos. Además, el procedimiento de aislamiento se puede repetir con el mismo frasco de cultivo por más de dos semanas. Este método podría ser aplicable a otras especies de mamíferos.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> Pentobarbital sódico solución </td><td> Kyoritsu Seiyaku </td><td> Somnopentyl inyección </td><td></td></tr><tr><td> Solución salina balanceada de Hank (HBSS) </td><td> Invitrogen </td><td> 14185-052 </td><td></td></tr><tr><td> Etilenglicol tetraacético (EGTA) </td><td> Sigma-Aldrich </td><td> E-4378 </td><td></td></tr><tr><td> Colagenasa </td><td> Wako Pure Chemical Industries, Ltd. </td><td> 032-10534 </td><td> Grado de disociación celular (Lote comprobar es necesario) </td></tr><tr><td> Inhibidor de la tripsina </td><td> Sigma-Aldrich </td><td> T-9128 </td><td></td></tr><tr><td> Medio esencial mínimo de Eagle (MEM) </td><td> Sigma-Aldrich </td><td> M-4655 </td><td></td></tr><tr><td> Dulbecco modificado Medio de Eagle (DMEM) </td><td> Sigma-Aldrich </td><td> D-6459 </td><td> Altos de glucosa de tipo </td></tr><tr><td> De suero fetal bovino (FCS) </td><td> HyClone </td><td> SH30070.03 </td><td> Inactivado por calor a 56 ° C durante 30 min. (Lote comprobar es necesario) </td></tr><tr><td> TrypLE expreso </td><td> Invitrogen </td><td> 12605 </td><td></td></tr><tr><td> Fosfato de Dulbecco (PBS) </td><td> Invitrogen </td><td> 14190 </td><td> Ca 2 +-Mg 2 + libre </td></tr><tr><td> β-mercaptoetanol </td><td> Sigma-Aldrich </td><td> M-7522 </td><td> Stock: 100 mM en agua destilada </td></tr><tr><td> Insulina </td><td> Sigma-Aldrich </td><td> I-5500 </td><td> Stock: 10 mg / ml de HCl 0,1 N </td></tr><tr><td> Penicilina / estreptomicina </td><td> Invitrogen </td><td> 15070 </td><td></td></tr><tr><td> Células colador </td><td> BD Biosciences </td><td> 352360 </td><td> Tamaño de la malla: 100 micras </td></tr><tr><td> Frasco de cultivo de tejidos </td><td> Sumitomo Bakelite Co., Ltd. </td><td> MS-21250 </td><td> Superficie: 75 cm 2 </td></tr><tr><td> Tejido no-cultura de plástico platos </td><td> BD Biosciences </td><td> 351005 </td><td></td></tr><tr><td> Raspador de células </td><td> Corning Inc. </td><td> 3010 </td><td></td></tr><tr><td> Recíproca agitador </td><td> TAITEC </td><td> NR-1 </td><td></td></tr></tbody></table>

a simple and efficient method to isolate macrophages from mixed primary cultures of adult liver cells

Las células de Kupffer son el hígado específicos de los macrófagos residentes y juegan un papel importante en las funciones fisiológicas y patológicas del hígado 1-3. Aunque los métodos de aislamiento de los macrófagos del hígado han sido bien descritos por 4-6, la mayor parte de estos métodos requiere de equipo sofisticado, como un elutriador centrífuga y habilidades técnicas. En este sentido, proporcionar un nuevo método para obtener los macrófagos del hígado, en cantidad y pureza de la mezcla de cultivos primarios de células de hígado de rata adulta, como se ilustra esquemáticamente en la Figura 1. Después de la disociación de las células del hígado en dos etapas método de perfusión 7,8, una fracción compuesta principalmente por los hepatocitos del parénquima se prepara y se sembró en frascos T75 de cultivo de tejidos con medio de cultivo compuesto por DMEM y 10% de los hepatocitos FCS.Parenchymal perder la morfología de las células epiteliales dentro de unos días en la cultura, degenerada o transformarse en células similares a fibroblastos (Figura 2). A medida que avanza la cultura, alrededor del día 6, de contraste de fase brillante, redondo como los macrófagos-células comienzan a proliferar en la lámina de células fibroblastos (Figura 2). El crecimiento de las células como los macrófagos-continuar y llegar a los niveles máximos alrededor del día 12, que cubre la capa celular en la superficie del frasco. Por agitación de los frascos de cultivo, los macrófagos son fácilmente suspendidas en el medio de cultivo. Posterior traslado e incubación corto en el resultado platos de plástico en la adhesión selectiva de los macrófagos (Figura 3), donde, como otras células contaminantes permanecen suspendidas. Después de varios lavados con PBS, los macrófagos adjunta se recogen. Más de 10 6 células pueden ser cosechadas varias veces de la misma T75 frasco de cultivo de tejidos de dos a intervalos de tres días por más de dos semanas (Figura 3). Las purezas de los macrófagos aislados de 95 a 99%, tal como se evaluó por citometría de flujo o inmunocitoquímica con ratas macrófagos anticuerpos específicos (Figura 4). Las células aisladas muestran fagocitosis activa de cuentas polystylene (Figura 5), ​​la respuesta proliferativa a GM-CSF recombinante, la secreción de antiinflamatorios / anti-citoquinas inflamatorias a la estimulación con LPS, y la formación de células gigantes multinucleadas 9. En conclusión, nos proporcionan un método simple y eficiente para obtener los macrófagos del hígado, en cantidad y pureza, sin un equipo complejo y skills.This método podría ser aplicable a otras especies de mamíferos.

Watch this Scientific Journal Video about A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells at JoVE.com

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

Un nuevo método para obtener los macrófagos de los cultivos primarios de células de hígado de rata se describe. Este método utiliza la proliferación de los macrófagos en la cultura, seguida de temblor en frascos de cultivo y la purificación por el apego selectivo para platos de plástico. Esta técnica proporciona de manera eficiente los macrófagos del hígado sin necesidad de equipo y habilidades complejas.

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 ...

a-simple-efficient-method-to-isolate-macrophages-from-mixed-primary

Research

JoVE Journal

Immunology and Infection

37.9K vistas.  National Institute of Agrobiological Sciences, Tsukuba, Japan. Un nuevo método para obtener los macrófagos de los cultivos primarios de células de hígado de rata se describe. Este método utiliza la proliferación de los macrófagos en la cultura, seguida de temblor en frascos de cultivo y la purificación por el apego selectivo para platos de plástico. Esta técnica proporciona de manera eficiente los macrófagos del hígado sin necesidad de equipo y habilidades complejas.

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

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 ...

Investigación

Inmunología e Infección

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

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

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

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

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

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

Un método simple y eficiente para detectar la activación del factor nuclear en neutrófilos humanos por citometría de flujo

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

La transfección altamente eficiente de humanos THP-1 macrófagos por Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Un método de rendimiento eficiente y de alta para el aislamiento de ratón subconjuntos de células dendríticas

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Un enfoque simple y eficiente de construcción de mutantes de virus vaccinia Vectores

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Generación e identificación de GM-CSF Derivado alveolares-como los macrófagos y las células dendríticas A partir de médula ósea de ratones

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

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 ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

Una forma no invasiva de aislar y células fenotípicas de la conjuntiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

Un método eficiente y Simple para establecer líneas de células T de pacientes con infección crónica activa Virus Epstein - Barr y NK

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...