Este trabajo fue apoyado por el CNRS (PICS 2012-2014 a EG) y por una beca de la Regione Campania (n.5 LR, 28.03.2002 a Giovanna Mottola). Filippo Conti es un compañero de la Cooperación Científica Fundación&#39;&#39; Infectiopole Sud&#39;&#39;. Nicola Boucherit es becario del Ministerio francés de Investigación y Tecnología. Las fuentes de financiación no tenía papel en el diseño del estudio, la recogida de datos, análisis de datos, decisión a publicar, o la preparación del manuscrito.

Declaración de Ética El protocolo para el manejo de animales fue aprobado por el Comité de Ética institucional Animal &quot;Conseil Scientifique du Centre de Formation et de Recherche Experimental Médico-Quirúrgica&quot; (CFREMC, permiso Proyecto 10-300122013 a Eric Ghigo) de la Universidad de Aix-Marsella, en acuerdo con las reglas del Decreto N ° 87-848 del 10/19/1987. Los experimentos se realizaron en la Faculté de Médecine de la Timone (número de permiso de Experimentación 13.385 a Eric Ghigo). 1. Material y Cultura Preparación de Medios <ol><li> Esterilizar dos pinzas, dos tijeras, dos cuchillas quirúrgicas, un mortero y una mano de mortero. </li><li> Obtener DMEM completo que contiene 10% de suero de ternera fetal (FCS), glutamina 2 mM, 100 U / ml de penicilina y 100 mg / ml de estreptomicina. </li><li> Diluir 10 veces en PBS agua destilada estéril para obtener PBS 1x. </li><li> Obtener enfriado con hielo 1x PBS, DMEM completo enfriado con hielo, WA DMEM completormado a 37 ° C. </li></ol> 2. Preparación de sobrenadantes de células L929 <ol><li> Crecer células L929 hasta confluencia (veinte por 175 cm 2 frascos) en DMEM completo a 37 ° C, 5% de CO 2. </li></ol> Nota: Se requiere de colonias de granulocitos y macrófagos factor estimulante (GM-CSF) para inducir la diferenciación de células hematopoyéticas en macrófagos 12. L929 células producen GM-CSF. <ol start="2"><li> En la confluencia, sustituir el medio de cultivo por DMEM completo fresco. Transferencia frascos a 32 ° C, 5% de CO 2 durante 10 días. </li><li> Recopilar, piscina y centrifugar los sobrenadantes a 750 g durante 10 min. Desechar los sedimentos celulares. </li><li> Sobrenadantes tienda en tubos de 15 ml y almacenar a -20 ° C. </li></ol> 3. Médula ósea derivada de macrófagos (BMDM) Preparación <ol><li> Sacrifica 1 ratón por dislocación cervical. <ol><li> Utilice cuchillas quirúrgicas estériles durante todo el experimento. Desinfecte la piel con 70% alcohol. Hacer una incisión en la parte superior de cada pata trasera y tire de la piel hacia abajo hacia el pie para exponer el músculo. </li><li> Cortar las patas traseras, quitar la piel con unas tijeras estériles y pinzas estériles. Coloque las piernas dentro de una placa de Petri estéril (35/10 mm) que contiene estéril, frío de hielo 1x PBS (5 ml). </li><li> Retire la carne y los músculos que se ha adherido a los huesos con tijeras y pinzas estériles. </li></ol></li><li> Transferencia de los huesos en una nueva, placa de Petri estéril (35/10 mm) que contiene enfriado con hielo, 1 x PBS estéril (5 ml). Lavar los huesos dos veces con 5 ml enfriado en hielo, 1x PBS estéril. </li><li> Transferir el hueso en un mortero estéril que contiene 5 ml enfriado en hielo, 1x PBS estéril. </li><li> Cortar la tibia del fémur en la articulación con tijeras estériles. Aplasta los huesos suavemente en un mortero estéril que contiene 5 ml enfriado en hielo, 1x PBS estéril usando una mano de mortero. </li><li> Recoger el sobrenadante en tubos de 15 ml enfriado en hielo. Repita este paso 3x. </li><li> Filtrar con un 70 micras stra celular NylonINER para eliminar los fragmentos sólidos. Centrifugar el filtrado a 450 xg durante 10 min a 4 ° C. </li><li> Deseche con cuidado el sobrenadante. Disociar el precipitado en 10 ml de sangre roja tampón de lisis celular para 30 seg. Añadir 20 ml enfriado en hielo, DMEM completo. </li></ol> Nota: El objetivo de este paso es eliminar la contaminación de las células rojas de la sangre, por lo tanto, este paso debe realizarse dentro de 2 min para evitar la alteración de células hematopoyéticas por la sangre tampón de lisis de glóbulos rojos. <ol start="8"><li> Se centrifuga a 450 × g durante 10 min a 4 ° C. Deseche con cuidado el sobrenadante. Disociar el precipitado en 20 ml de DMEM completo que se ha calentado a 37 ° C. </li><li> Transferencia de las células disociadas en 2 placas de Petri (100/20 mm). Incubar durante 4 horas a 37 ° C. </li><li> Reunir los sobrenadantes en tubos de 50 ml a temperatura ambiente. Deseche los platos que contienen los macrófagos residentes. </li></ol> Nota: El objetivo de este paso es eliminar el marro hueso residentemacrófagos w por su capacidad de adherirse a plástico de cultivo tratados. Estos macrófagos residentes pueden ser utilizados en otros experimentos. <ol start="11"><li> Centrifugar los sobrenadantes recogidos a 450 xg durante 10 min a 4 ° C. Eliminar el sobrenadante. </li><li> Disociar suavemente el sedimento en 10 ml de DMEM completo que contiene 15% de L929 sobrenadante celular. Filtre las células a través de una 40 m de células colador de nylon. </li><li> Recuperar el filtrado. Añadir el filtrado recogido (10 ml) a 140 ml de DMEM completo que se ha suplementado con 15% de L929 medio celular. </li><li> Distribuir 10 ml de la suspensión de células por placa de Petri (15 placas de Petri, 100/20 mm). Se incuban las células a 37 ° C, 5% de CO 2. </li><li> Se cultivan las células durante 3 días </li><li> Añadir 10 ml de DMEM completo que se ha suplementado con 15% de L929. Se incuban las células durante 4 días adicionales. </li></ol> Nota: Supervisar periódicamente el crecimiento de células con un microscopio invertido (pasos 3.14 a 3.16). Macrófagos adherentes seránobservado después de 3 días de cultivo. 4. BMDMs Cosecha <ol><li> Eliminar el sobrenadante. Lave BMDMs 2 veces con DMEM completo </li><li> Añadir 5 ml de DMEM completo que se ha calentado a 37 ° C. Separe BMDMs raspando suavemente con una espátula de caucho. </li><li> Recoger BMDMs en tubos de 50 ml y centrifugar a 450 xg durante 10 min. Disociar suavemente sedimentos celulares en 20 ml de DMEM completo. </li><li> Cuente BMDMs en presencia de azul de tripano (sin mortalidad superior al 10% debe ser observado). </li></ol> Nota: Por lo general, alrededor de 6-7,5 x 10 7 macrófagos se obtuvieron a partir de 15 placas de Petri (100/20 mm) de los macrófagos. Hay aproximadamente 4-5 x 10 6 macrófagos / placa de Petri (20/100 mm). <ol start="5"><li> Preparar BMDMs según sea necesario para el experimento. Después de 16 h en medio completo a 37 ° C, 5% de CO 2, macrófagos volverá a adherirse al soporte. </li></ol> 5. Almacenamiento BMDMs <ol><li> Recoger BMDMs aislados (paso 4.3). Se centrifuga a 450 × g durante 10 min. Nota: BMDMs pueden congelarse en nitrógeno líquido. </li><li> Resuspender los sedimentos celulares en medio de congelación que consiste en 10% de DMSO y 90% de FCS a una concentración final de 4 x 10 6 células / ml y pipeta de 1 ml en cada ampolla. </li><li> Congelar las células a una velocidad de enfriamiento de 1 º C / min. Después de 24 horas, transferir la ampolla a un contenedor de nitrógeno líquido para almacenamiento a largo plazo. </li></ol>

<ol>
	<li>Rutherford, M. S., Witsell, A., Schook, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+generating+functionally+heterogeneous+macrophages:+chaos+revisited.">Mechanisms generating functionally heterogeneous macrophages: chaos revisited.</a> J. Leukocyte Biol. 53, 602-618 (1993).</li><li>Van Furth, R., Gallin, J. I., Goldstein, I. M., Snyderman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Inflammation: Basic Principles and Clinical Correlates. 112, 325-336 (1992).</li><li>Adams, D., Halmiton, T., Gallin, J. I., Goldstein, I. M., Snyderman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Inflammation: Basic Principles and Clinical Correlates. 112, 325-336 (1992).</li><li>Morris, L., Graham, C. F., Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+in+haemopoietic+and+other+tissues+of+the+developing+mouse+detected+by+the+monoclonal+antibody+F4/80.">Macrophages in haemopoietic and other tissues of the developing mouse detected by the monoclonal antibody F4/80.</a> Development. 112, 517-526 (1991).</li><li>Haas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phagosome:+compartment+with+a+license+to+kill.">The phagosome: compartment with a license to kill.</a> Traffic. 8, 311-330 (2007).</li><li>Underhill, D. M., Ozinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+of+microbes:+complexity+in+action.">Phagocytosis of microbes: complexity in action.</a> Annu. Rev. Immunol. 20, 825-852 (2002).</li><li>Castagna, A., Polati, R., Bossi, A. M., Girelli, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte/macrophage+proteomics:+recent+findings+and+biomedical+applications.">Monocyte/macrophage proteomics: recent findings and biomedical applications.</a> Expert Rev. Proteomics. 9, 201-215 (2012).</li><li>Benoit, M., Desnues, B., Mege, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+polarization+in+bacterial+infections.">Macrophage polarization in bacterial infections.</a> J. Immunol. 181, 3733-3739 (2008).</li><li>Barry, A. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+stimulation+of+p38alpha-MAPK/Vps41-HOPS+by+LPS+from+pathogenic+Coxiella+burnetii+prevents+trafficking+to+microbicidal+phagolysosomes.">Impaired stimulation of p38alpha-MAPK/Vps41-HOPS by LPS from pathogenic Coxiella burnetii prevents trafficking to microbicidal phagolysosomes.</a> Cell Host Microbe. 12, 751-763 (2012).</li><li>Henry, R. M., Hoppe, A. D., Joshi, N., Swanson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+uniformity+of+phagosome+maturation+in+macrophages.">The uniformity of phagosome maturation in macrophages.</a> J. Cell Biol. 164, 185-194 (2004).</li><li>Sundaramurthy, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+Chemical+and+RNAi+Multiparametric+Profiles+Identifies+Triggers+of+Intracellular+Mycobacterial+Killing.">Integration of Chemical and RNAi Multiparametric Profiles Identifies Triggers of Intracellular Mycobacterial Killing.</a> Cell Host Microbe. 13, 129-142 (2013).</li><li>Boltz-Nitulescu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+rat+bone+marrow+cells+into+macrophages+under+the+influence+of+mouse+L929+cell+supernatant.">Differentiation of rat bone marrow cells into macrophages under the influence of mouse L929 cell supernatant.</a> J. Leukocyte Biol. 41, 83-91 (1987).</li></ol>

No hay conflictos de interés declarado.

El protocolo descrito en el presente documento detalla un método para producir un gran número de BMDMs. BMDM son células primarias y tienen la función y las propiedades de los macrófagos a partir de monocitos diferenciados biológica porque hay madura, en contraste a las líneas celulares de macrófagos, que son inmaduros. BMDMs pueden ser utilizados para la investigación genética (RNAi), la detección de drogas, los estudios funcionales, estudios de interacción huésped-patógeno y muchas otras áreas de invest...

Un aspecto significativo de la función de los macrófagos es su papel en la inmunidad innata y adaptativa. Debido a su capacidad de fagocitar partículas inertes, bacterias o parásitos, los macrófagos son una primera línea de defensa contra los invasores extranjeros. Una vez interiorizado, los microbios son degradados en los fagolisosomas. Los macrófagos también envían señales de contratación para y presentan antígenos a otras células inmunes, como los linfocitos T. Los macrófagos se derivan de monocitos. Los monocitos se originan en la médula ósea a partir de células madre mieloides y migran a la sangre periférica y diversos tejidos donde se diferencian en macrófagos. Se estima que un ratón adulto sano contiene aproximadamente 10 8 macrófagos que se distribuyen por todo el cuerpo en varios órganos y tejidos (Tabla 1) 1,2. Los macrófagos muestran una gran diversidad fenotípica y funcional debido a su capacidad de adaptarse a su 3,4 microambiente. El macrop más importanteHage propiedad es su actividad microbicida, que se define por su capacidad para fagocitar y destruir microbios ellos. La respuesta fagocítica se define por la activación de complejas redes de señalización que son estimulados por el contacto microbiana, por lo tanto, los macrófagos modulan la expresión génica apropiada en respuesta a diversos estímulos. Después de la fagocitosis, los microbios se eliminan en una estructura llamada fagolisosoma, sin embargo, muchos microbios patógenos han desarrollado estrategias para subvertir la función microbicida de los macrófagos 5. La diversidad de mecanismos de subversión que son utilizados por diferentes especies microbianas es un testimonio de la complejidad del proceso de fagocitosis 6 y fagolisosoma biogénesis. Las enfermedades infecciosas son importantes problemas de salud humana, y numerosos mecanismos y moléculas participan en la actividad antimicrobiana de los macrófagos. Además, los objetivos de las propiedades microbicidas que son secuestrados por los microbios siguen siendo desconocidos, por lo que no es un correoxplosion de interés en los aspectos evolutivos, genéticos y bioquímicos de las interacciones huésped-patógeno que ha renovado la atención científica en relación con los macrófagos. En la actualidad, la mayoría de la investigación en el campo se hace en macrófagos líneas celulares, que difieren de los macrófagos primarios en la actividad fagocítica, la producción de citoquinas y la regulación de la explosión oxidativa. Además, son menos adecuados para la microscopía. Para investigar la interacción macrófagos-patógenos, se recomienda utilizar los macrófagos primarios, como la médula de macrófagos derivados (BMDMs), que presentan características más fisiológicas. Además, es posible trabajar en BMDMs modificados genéticamente, debido a que estos macrófagos pueden ser aislados directamente a partir de ratones transgénicos y, con la disponibilidad de nuevas tecnologías, tales como la transfección lentiviral, su perfil de expresión génica puede ser alterada por la sobreexpresión del gen o ARN de interferencia. Aquí se describe un procedimiento para diferenciar murino m huesoflecha en macrófagos que proporcionarán un gran número de macrófagos en 7 días para diversos análisis funcional, como la proteómica 7, 8 transcriptómica, tráfico intracelular estudia 9, 10 estudios dinámicos, pantallas genéticas (RNAi), y detección de drogas 11.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco Life Technologies</td>
			<td>21969-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Gibco Life Technologies</td>
			<td>10270</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco Life Technologies</td>
			<td>15070</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Gibco Life Technologies</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10x)</td>
			<td>Lonza</td>
			<td>BEM515F</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis buffer</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 70 &#956;m Nylon</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 40 &#956;m Nylon</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes (100/20 mm)</td>
			<td>any supplier</td>
			<td>n/a</td>
			<td>culture treated</td>
		</tr>
		<tr>
			<td>Petri dishes (35/10 mm)</td>
			<td>any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

bone marrow-derived macrophage production

Los macrófagos son componentes fundamentales de la respuesta inmune innata y adaptativa, y son la primera línea de defensa contra los invasores extranjeros debido a sus actividades microbicidas poderosos. Los macrófagos se distribuyen ampliamente por todo el cuerpo y están presentes en los órganos linfoides, hígado, pulmones, tracto gastrointestinal, sistema nervioso central, hueso, y la piel. Debido a su reparto, que participan en una amplia gama de procesos fisiológicos y patológicos. Los macrófagos son células altamente versátiles que son capaces de reconocer alteraciones microambientales y para mantener la homeostasis del tejido. Numerosos patógenos han desarrollado mecanismos para utilizar los macrófagos como caballos de Troya para sobrevivir, replicarse en, e infectar a los seres humanos y los animales y de propagar por todo el cuerpo. La reciente explosión de interés en los aspectos evolutivos, genéticos y bioquímicos de las interacciones huésped-patógeno ha renovado la atención científica en relación con los macrófagos. Aquí se describe unprocedimiento para aislar y cultivar los macrófagos de la médula ósea murina que proporcionará un gran número de macrófagos para el estudio de las interacciones huésped-patógeno, así como otros procesos.

El objetivo de este método era obtener fácilmente un gran número de macrófagos en pocos días. La preparación de células de médula ósea se ilustra en la Figura 1. Los huesos de la pata trasera se recogieron y se rompieron en un mortero. Una vez que los macrófagos residentes fueron retirados de la preparación de células de médula ósea, células de médula ósea se incubaron con GM-CSF (Día 0). Después de 3 días, las células, que eran redondas y no adherente antes del cultivo, comienzan a...

Watch this Scientific Journal Video about Bone Marrow-derived Macrophage Production at JoVE.com

Bone Marrow-derived Macrophage Production

Los macrófagos han sido reconocidos como un componente crítico de la respuesta inmune innata y adaptativa. La reciente explosión de conocimientos relativos a los aspectos evolutivos, genéticos y bioquímicos de la interacción entre los macrófagos y los microbios ha renovado la atención científica a los macrófagos. Este artículo describe un método para diferenciar los macrófagos de médula ósea de ratón.

Médula ósea derivada de macrófagos Producción

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

bone-marrow-derived-macrophage-production

Research

JoVE Journal

Immunology and Infection

72.8K vistas.  Aix-Marseille Université.  Los macrófagos han sido reconocidos como un componente crítico de la respuesta inmune innata y adaptativa. La reciente explosión de conocimientos relativos a los aspectos evolutivos, genéticos y bioquímicos de la interacción entre los macrófagos y los microbios ha renovado la atención científica a los macrófagos. Este artículo describe un método para diferenciar los macrófagos de médula ósea de ratón. 

Video: Bone Marrow-derived Macrophage Production

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

Quiste Toxoplasma gondii Muro de Formación en Activado médula ósea procedentes de los macrófagos y Condiciones bradizoíto

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

Investigación

Inmunología e Infección

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

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

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

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

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

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

Investigación de la polarización de macrófagos utilizando la médula ósea macrófagos derivados

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

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 ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

Caracterización metabólica de polarizado M1 y macrófagos derivados de Médula ósea M2 Uso del análisis extracelular Flux en tiempo real

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Evaluación de los efectos de drogas basados en anticuerpos en médula ósea murina y activación de los macrófagos peritoneales

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Detección de Inflammasome activación y muerte celular Pyroptotic en macrófagos derivados de médula ósea murinas

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

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

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

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

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

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

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

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

This protocol has been established to culture tetrahydrobiopterin (BH4)- and inducible nitric oxide synthase (iNOS)-deficient primary murine macrophages to study NO-redox biology. The study focuses on reducing potential contamination of BH4 and other artifacts found in traditional isolation and culture methods which may confound experimental outcomes and interpretation of results.

Aislamiento y cultivo in vitro de macrófagos derivados de la médula ósea para el estudio de la biología NO-redox

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

Protocolo económico y eficiente para aislar y cultivar células dendríticas derivadas de la médula ósea de ratones

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...