Este trabalho foi apoiado pelo CNRS (PICS 2012-2014 a GE) e por uma bolsa da Regione Campania (LR n.5, 28.03.2002 para Giovanna Mottola). Filippo Conti é membro da Cooperação Científica Foundation&#39;&#39; Infectiopole Sud.&#39;&#39; Nicola Boucherit é um companheiro do Ministério Francês de Pesquisa e Tecnologia. As fontes de financiamento não teve nenhum papel no desenho do estudo, coleta de dados, análise de dados, a decisão de publicar ou preparação do manuscrito.

Declaração de Ética O protocolo para manejo dos animais foi aprovado pelo Comitê de Ética institucional nosso Animal &quot;Conseil Scientifique du Centre de Formation et de Recherche Experimental Médico-Cirúrgica&quot; (CFREMC, licença Projeto 10-300122013 para Eric Ghigo) da Universidade de Aix-Marseille de acordo com as regras Décret de N ° 87-848 de 1987/10/19. Os experimentos foram realizados na Faculté de Médecine de la Timone (número de autorização Experimentação 13,385 para Eric Ghigo). 1. Material e Meios de Cultura Preparação <ol><li> Esterilizar duas pinças, duas tesouras, duas lâminas cirúrgicas, um almofariz e um pilão. </li><li> Obter DMEM completo contendo 10% de soro fetal de vitelo (FCS), 2 mM de glutamina, 100 U / ml de penicilina e 100 ug / ml de estreptomicina. </li><li> Diluir 10x PBS em água destilada estéril para obter 1x PBS. </li><li> Obter gelada PBS 1x, gelada DMEM completo, wa DMEM completormed a 37 ° C. </li></ol> 2. Preparação de sobrenadantes de células L929 <ol><li> Crescer as células L929 até à confluência (vinte e dois frascos de 175 centímetros) em DMEM completo a 37 ° C, 5% de CO 2. </li></ol> Nota: granulócitos-macrófagos colónia factor (GM-CSF) estimulante é necessária para induzir a diferenciação de células hematopoiéticas em macrófagos 12. L929 células produzem GM-CSF. <ol start="2"><li> Na confluência, substituir meios de cultura com DMEM completo fresco. Transferir para frascos de 32 ° C, 5% de CO 2 durante 10 dias. </li><li> Coletar, piscina e centrifugar o sobrenadante a 750 g durante 10 min. Descartar sedimentos celulares. </li><li> Sobrenadantes Armazene em tubos de 15 ml e armazenar a -20 ° C. </li></ol> 3. Bone Marrow-derivado do macrófago (BMDM) Preparação <ol><li> Sacrifique um rato por deslocamento cervical. <ol><li> Use lâminas cirúrgicas estéreis durante todo o experimento. Desinfetar a pele com 70% alcohol. Realizar uma incisão na parte superior de cada perna traseira e puxe a pele para baixo em direcção ao pé para expor o músculo. </li><li> Corte as pernas traseiras, retire a pele com uma tesoura estéril e pinças esterilizadas. Coloque as pernas numa placa de Petri estéril (35/10 mm) contendo estéril gelada 1x PBS (5 ml). </li><li> Retire a carne e músculos que estão aderindo aos ossos com tesouras e pinças esterilizadas. </li></ol></li><li> Transfira os ossos para uma nova placa de Petri estéril (35/10 mm) contendo gelado, estéril 1x PBS (5 ml). Lave os ossos duas vezes com 5 ml de gelado, estéril PBS 1x. </li><li> Transferir o osso em um almofariz estéril contendo 5 ml de gelado, estéril PBS 1x. </li><li> Corte a tíbia do fêmur na articulação com tesoura esterilizada. Esmagar os ossos suavemente num almofariz estéril contendo 5 ml de gelado, estéril 1x PBS utilizando um pilão. </li><li> Recolhe-se o sobrenadante em tubos de 15 ml de gelo-frio. Repita este passo 3x. </li><li> Filtrar com um 70 um stra célula NylonIner para remover fragmentos sólidos. Centrifuga-se o filtrado a 450 xg durante 10 min a 4 ° C. </li><li> Delicadamente, desprezar o sobrenadante. Dissociar o sedimento em 10 ml de tampão de lise de glóbulos vermelhos para 30 seg. Adicionar 20 ml de gelado, DMEM completo. </li></ol> Nota: O objectivo deste passo é o de remover contaminantes de células vermelhas do sangue, portanto, este passo deve ser realizado no prazo de 2 minutos, para evitar a alteração de células hematopoiéticas por o tampão de lise de glóbulos vermelhos. <ol start="8"><li> Centrifugar a 450 xg durante 10 min a 4 ° C. Delicadamente, desprezar o sobrenadante. Dissociar o sedimento em 20 ml de DMEM completo que foi aquecido a 37 ° C. </li><li> Transferir as células dissociadas em duas placas de Petri (100/20 mm). Incubar durante 4 horas a 37 ° C. </li><li> Recolhe-se os sobrenadantes em tubos de 50 ml à temperatura ambiente. Rejeitar os pratos que contêm os macrófagos residentes. </li></ol> Nota: O objetivo deste passo é eliminar o marro osso residentemacrófagos w por sua capacidade de aderir ao plástico tratados com cultura. Estes macrófagos residentes podem ser usados ​​em outras experiências. <ol start="11"><li> Centrifugar os sobrenadantes recolhidos a 450 xg durante 10 min a 4 ° C. Desprezar o sobrenadante. </li><li> Dissociar suavemente o sedimento em 10 ml de DMEM completo contendo 15% de sobrenadante de células L929. Filtrar as células através de um coador de células de 40 mM de nylon. </li><li> Recuperar o filtrado. Adicionar o filtrado recolhido (10 ml) a 140 ml de DMEM completo que foi suplementado com 15% de meios de células L929. </li><li> Distribuir 10 ml da suspensão de células por placa de Petri (15 placas de Petri, 100/20 mm). Incubar as células a 37 ° C, 5% de CO 2. </li><li> Crescer as células durante 3 dias </li><li> Adicionar 10 ml de DMEM completo que foi suplementado com 15% L929. Incubar as células durante 4 dias adicionais. </li></ol> Nota: Monitorar periodicamente o crescimento de células com um microscópio invertido (passos 3,14-3,16). Macrófagos aderentes seráobservado após 3 dias de cultura. 4. BMDMs Colheita <ol><li> Remover o sobrenadante. Lavar BMDMs 2 vezes com DMEM completo </li><li> Adicionar 5 ml de DMEM completo que foi aquecido a 37 ° C. Retire BMDMs raspando suavemente com um policial de borracha. </li><li> Recolhe BMDMs em tubos de 50 ml e centrifugar a 450 xg durante 10 min. Suavemente dissociar os sedimentos celulares em 20 ml de DMEM completo. </li><li> Contagem BMDMs na presença de azul de tripano (não houve mortalidade de mais de 10% devem ser observados). </li></ol> Nota: Em geral, cerca de 6-7,5 x 10 7 macrófagos são obtidos a partir de 15 placas de Petri (100/20 mm) dos macrófagos. Há cerca de 4-5 x 10 6 macrófagos / placa de Petri (100/20 mm). <ol start="5"><li> Prepare BMDMs conforme necessário para o experimento. Depois de 16 horas em meio completo a 37 ° C, 5% de CO 2, macrófagos vai novamente aderir ao suporte. </li></ol> 5. Armazenar BMDMs <ol><li> Colete BMDMs isolados (passo 4.3). Centrifugar a 450 xg durante 10 min. Nota: BMDMs podem ser congeladas em nitrogênio líquido. </li><li> Os sedimentos de células ressuspender em meio de congelação consistindo em 10% de DMSO e 90% de FCS a uma concentração final de 4 x 10 6 células / ml e pipeta de 1 ml em cada ampola. </li><li> Congelar as células a uma taxa de arrefecimento de 1 ° C / min. Após 24 horas, transferir a ampola a um contentor de azoto líquido para o armazenamento a longo prazo. </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>

Não há conflitos de interesse declarados.

O protocolo aqui descrito detalha um método para produzir grandes números de BMDMs. BMDM são células primárias e têm a função biológica e as propriedades dos macrófagos diferenciados a partir de monócitos, porque há madura, em contraste com as linhas celulares de macrófagos, que são imaturos. BMDMs podem ser utilizados para o rastreio genético (RNAi), a triagem de drogas, estudos funcionais, estudos de interação patógeno-hospedeiro e muitas outras áreas de investigação. ...

Um aspecto importante da função dos macrófagos é o seu papel na imunidade inata e adaptativa. Devido à sua capacidade de fagocitar partículas inertes, bactérias ou parasitas, os macrófagos são a primeira linha de defesa contra os invasores externos. Uma vez internalizado, micróbios são degradados dentro de fagolisossomas. Os macrófagos também enviam sinais de recrutamento para e antígenos presentes a outras células do sistema imunológico, como os linfócitos T. Os macrófagos são derivados dos monócitos. Os monócitos surgem na medula óssea a partir de células-tronco mielóides e migrar para o sangue periférico e vários tecidos, onde eles diferenciam-se em macrófagos. Estima-se que um rato adulto saudável contém aproximadamente 10 8 macrófagos que são distribuídos ao longo do corpo em vários órgãos e tecidos (Tabela 1) 1,2. Os macrófagos exibem grande diversidade fenotípica e funcional por causa de sua capacidade de se adaptar ao seu microambiente 3,4. O mais importante macrophage propriedade é a sua actividade microbicida, a qual é definida pela sua capacidade de fagocitose de micróbios e as destroem. A resposta fagocítica está definido pela activação de redes de sinalização complexas que são estimulados por contacto microbiana, assim, para modular a expressão de genes de macrófagos de forma apropriada em resposta a diversos estímulos. Após a fagocitose, os micróbios são eliminados em uma estrutura chamada fagolisossomo, no entanto, muitos micróbios patogénicos têm desenvolvido estratégias para subverter a função microbicida dos macrófagos 5. A diversidade de mecanismos de subversão que são utilizadas por diferentes espécies microbianas é uma prova da complexidade do processo de fagocitose 6 e fagolisossomo biogênese. As doenças infecciosas são os principais problemas de saúde humana, e vários mecanismos e moléculas de participar de atividades antimicrobianas de macrófagos. Além disso, os alvos das propriedades microbicidas que são sequestradas por micróbios permanecem desconhecidos, assim, existe uma mensagemxplosion de interesse nos aspectos evolutivos, genéticos e bioquímicos de interação patógeno-hospedeiro, que renovou a atenção científica sobre macrófagos. Atualmente, a maioria das pesquisas no campo é feito em macrófagos linhagens celulares, que diferem de macrófagos primários na atividade fagocitária, a produção de citocinas e da regulação do burst oxidativo. Além disso, eles são menos adequados para microscopia. Investigar a interação macrófagos-patógenos é recomendado o uso de macrófagos primários, tais como medula macrófagos derivados (BMDMs), que apresentam características mais fisiológicas. Além disso, é possível trabalhar em BMDMs geneticamente modificados, porque estes macrófagos pode ser isolado directamente a partir de ratinhos transgénicos e, com a disponibilidade de novas tecnologias, tais como a transfecção lentiviral, o perfil de expressão do gene podem ser alterados por sobre-expressão do gene ou ARN de interferência. Aqui, descrevemos um procedimento para diferenciar murino osso marrow em macrófagos que irão proporcionar um grande número de macrófagos em 7 dias para várias análises funcionais, como a proteômica 7, 8 transcriptômica, tráfico intracelular estuda 9, estudos dinâmicos 10, telas genéticos (RNAi) e triagem 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

Os macrófagos são componentes críticos das respostas imune inata e adaptativa, e eles são a primeira linha de defesa contra invasores estrangeiros por causa de suas poderosas atividades microbicidas. Os macrófagos são amplamente distribuído por todo o corpo e estão presentes nos órgãos linfóides, fígado, pulmões, tracto gastrointestinal, sistema nervoso central, ossos e pele. Por causa da sua repartição, que participam numa vasta gama de processos fisiológicos e patológicos. Os macrófagos são células altamente versáteis que são capazes de reconhecer alterações do microambiente e para manter a homeostase do tecido. Numerosos agentes patogénicos desenvolveram mecanismos para usar macrófagos como cavalos de Tróia para sobreviver, replicam em, e infectam os seres humanos e animais e para propagar ao longo do corpo. A recente explosão de interesse em aspectos evolutivos, genéticos e bioquímicos de interação patógeno-hospedeiro renovou atenção científica sobre macrófagos. Aqui, descrevemos umProcesso para isolar e cultivar macrófagos de medula óssea de murino que irá proporcionar um grande número de macrófagos para estudar interacções hospedeiro de organismos patogénicos, assim como outros processos.

O objectivo deste método é obter facilmente um grande número de macrófagos em poucos dias. A preparação de células da medula óssea é ilustrada na Figura 1. Ossos da perna foram coletadas e esmagado num almofariz. Uma vez que os macrófagos residentes foram removidos a partir da preparação de células de medula óssea, células da medula óssea foram incubadas com GM-CSF (Dia 0). Após 3 dias, as células, que foram rodada e não aderentes antes da cultura, começam a diferenciar-se em macróf...

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

Bone Marrow-derived Macrophage Production

Os macrófagos têm sido reconhecidos como um componente crítico das respostas imune inata e adaptativa. A recente explosão de conhecimentos relativos aos aspectos evolutivos, genéticos e bioquímicos da interação entre macrófagos e micróbios renovou atenção científica para macrófagos. Este artigo descreve um método para diferenciar macrófagos de medula óssea de ratinho.

Medula Óssea derivadas de macrófagos Produção

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 visualizações.  Aix-Marseille Université.  Os macrófagos têm sido reconhecidos como um componente crítico das respostas imune inata e adaptativa. A recente explosão de conhecimentos relativos aos aspectos evolutivos, genéticos e bioquímicos da interação entre macrófagos e micróbios renovou atenção científica para macrófagos. Este artigo descreve um método para diferenciar macrófagos de medula óssea de ratinho. 

Vídeo: 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.

Toxoplasma gondii Formação parede do cisto no Activated derivadas da medula óssea macrófagos e Condições Bradyzoite

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

Imunologia e Infecção

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.

Geração e Rotulagem de murino derivadas da medula óssea Células Dendríticas com Nanocristais Qdot de Estudos de rastreamento

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.

Investigação de polarização de macrófagos Usando medula 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).

Isolamento e injecção intravenosa de Murino de Medula Óssea Derivado monócitos

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.

Caracterização metabólica de Polarizada M1 e M2 macrófagos da medula óssea derivadas usando análises em tempo real do fluxo extracelular

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.

Avaliação dos efeitos de drogas baseados em anticorpo murino da medula óssea e ativação de macrófagos peritoneais

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.

Deteção de ativação de Inflammasome e morte celular de Pyroptotic nos macrófagos da medula óssea-derivado murino

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.

Um modelo de rato In Vivo para ativação de células T Naïve CD4 medida, proliferação e diferenciação de células Th1 induzida pelas células dendríticas derivadas de medula óssea

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.

Isolamento e Cultura In vitro de Macrófagos Derivados da Medula Óssea para o Estudo da Biologia 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 Econômico e Eficiente para Isolar e Culturing Células Dendríticas Derivadas da Medula Óssea de camundongos

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