Este trabalho foi financiado pelo NIEHS R01ES014470 (KMZ).

Antes de iniciar este protocolo, recolher os ratos neonatal nos dias pós-natal 1-3 (P1-3) em um recipiente estéril aninhados com roupa de cama gaiola original para proteção e calor. É importante trabalhar com rapidez e eficiência através deste protocolo para otimizar o rendimento da microglia. Por favor, consulte a Tabela 1 para a lista de reagentes completa. 1. Preparação de instrumentos, Meios de Cultura, e pratos <ol><li> Preparar 10 ml / filhote Microglia Meio Completo (MCM; 1x Essencial Mínimo de Meio Earle [MEM], suplementado com: 1 mM de L-glutamina, piruvato de sódio 1 mM, 0,6% v / v D-(+)-glucose, 100 ug / ml penicilina / estreptomicina (P / S), 4% v / v de soro fetal bovino (FBS), 6% v / v de soro de cavalo), e adicionar a cada frasco T-75 (um frasco / cão); tampa e horizontalmente em lugar uma incubadora (37 ° C, 5% CO 2) para se equilibrar durante 1 hora. É essencial para equilibrar este meio em frasco (s) antes de iniciar a dissecção. </li><li> Prepare, alíquota, e quente em 37 ° C MCM banho-maria por dissecção (6 ml / filhote de cachorro) e em um tubo cônico MCM separado para a ressuspensão celular (2 ml / filhote de cachorro). Preparar, alíquota, e frio em gelo trituração meios (MM; HBSS suplementado com: 100 mg / ml de P / S, 0,01 M HEPES, 3 ml / cão), e meios de dissecação (DM; MEM suplementado com 100 ug / ml de P / S; 2 ml / filhote de cachorro). </li><li> Esterilizar os seguintes instrumentos cirúrgicos por submersão em etanol 70% por 10 min: tesoura # 1 (1; decapitar), fórceps # 1 (2, para proteger o nariz e remover córtices), tesoura # 2 (1; nonangled; abrir couro cabeludo ), fórceps # 2 (1; remover couro cabeludo), fórceps # 3 (1, para remover o crânio), fórceps # 4 (2; remover meninges), tesoura # 3 (1; picar o tecido cerebral). Deixe secar ao ar em uma almofada estéril. </li><li> Limpe a estação de trabalho do fluxo de ar horizontal e dissecção microscópio com etanol 70%. </li><li> Dispensar DM em 2 estéreis, 100 milímetros placas de Petri para a captura de cabeça (3 ml, 1 prato / 3 filhotes) e dissecção (1 ml;1 prato / filhote; refrigerados em gelo antes da dissecação). Dispensar MM em um estéril, 100 milímetros placa de Petri para picar o tecido cerebral (1 ml, 1 prato / filhote de cachorro). </li></ol> 2. Tecido cerebral Dissecção <ol><li> Antes da eutanásia, higienizar suavemente pescoço e cabeça de cachorro com etanol 70%, utilizando um tecido de laboratório. Com tesoura # 1, eutanásia rapidamente filhote, removendo a cabeça, cabeça de captura previamente preparado em placa de Petri. Eutanásia e completar a dissecação de um filhote de cachorro antes de prosseguir para o próximo animal. </li><li> Transfira a cabeça para o prato dissecção refrigerados; sob microscópio de dissecação proteger a cabeça, apertando o nariz com pinça # 1 (em alternativa, a cabeça pode ser assegurada por piercing fórceps através do soquete do olho); couro cabeludo aberto usando tesoura º 2, criando um Y-cut: começar a partir da base da cabeça e em um ângulo em direção aos olhos. Use pinça n º 3 para remover suavemente couro cabeludo, descamação lateralmente, expondo o crânio. </li><li> Usando fórceps # 3, retire cuidadosamente tecido conjuntivo na base dacabeça. Quando a base do crânio é exposta crânio, cuidadosamente aberta enfiando um braço de pinça entre o cérebro eo crânio (após a fissura inter-hemisférica); aderência; puxe o crânio para cima para rasgar. </li><li> Disrupt tecido conjuntivo sob o lado ventral do cérebro usando pinça n º 1 e, posteriormente, remover cérebro usando a mesma ferramenta. Levante o cérebro fora do crânio, utilizando uma força para cima sob o lado ventral do cérebro. </li><li> Mantenha o cérebro em sua posição anatômica: Lado dorsal voltada para cima para visualizar os córtices sob o microscópio de dissecação; proteger o cérebro no lugar através da inserção de pontas de uma pinça na interseção do mesencéfalo eo córtex; remover completamente meninges corticais com pinça # 4, em seguida, proceder para remover as meninges ventral. Uma vez que as meninges foram completamente removidos, os córtices separar do resto do cérebro utilizando fórceps # 4. É essencial para remover completamente o tecido das meninges para maximizar a pureza e microglia yield. </li></ol> 3. Tecido Cerebral Picar <ol><li> Córtices Transferência para preparado previamente picados prato; continuar a picar passo usando técnica estéril na Classe II cabine de segurança biológica. </li><li> Tecido cerebral Mince usando tesoura # 3; transferência tecido picada de um tubo de 15 ml;. Lavar tesoura e picar prato com 1 ml de MM cada, recolher e distribuir lavagens para o tubo de 15 ml contendo o tecido picada Esta etapa de lavagem irá maximizar o rendimento da microglia, assegurando que todo o tecido residual é transferido para o tubo cónico. Deixe suspensão de tecido sem ser perturbado por 1-2 min, permitindo que o tecido picada para resolver na parte inferior do tubo. Não exceda 2 min como MM não contém nutrientes essenciais. </li><li> Cuidadosamente retire e descarte 2 ml de sobrenadante com genérico 1.000 mL micropipeta com uma ponteira standard, tornando certas para não perturbar o tecido picada resolvido. </li&gt; <li> Adicionar 3 ml de MCM para o tecido moído; tritura-se o tecido com força total usando um genérico 1000 ul micropipeta com uma ponta de pipeta padrão discernível n tecido sólido devem permanecer quando trituração é completa.. Adicionar mais 3 ml de MCM com a mesma ponteira, coletando amostra triturado residual da ponta da pipeta. </li></ol> 4. Crescer células corticais <ol><li> Centrífuga tecido picada para sedimentar as células (215 xg, 5 min; RT; centrífuga clínica com rotor basculante). Retornar células peletizadas para a cabine de segurança biológica, remover e descartar o sobrenadante; delicadamente ressuspender o pellet celular (2 ml / tubo; MCM); adicionar o ressuspensão celular para o T-75 frasco previamente preparado; tampar a garrafa e coloque-o horizontalmente em um humidificado, incubador padrão de cultura de tecidos (37 ° C, 5% CO 2). Permitir que as células a incubar durante 24 horas. </li><li> Após 24 horas, bata suavemente frasco contra a palma da mão para interromperrestos de tecido. Confira as células antes e depois de tocar para garantir a completa remoção de detritos. Conjunto de frasco vertical; remover e descartar media; adicionar MCM fresco para o fundo do frasco (12 ml; 37 ° C); tampa e volte a colocar na incubadora, horizontalmente. </li><li> Confira as células diariamente para a contaminação e para monitorar o crescimento. </li><li> Em cerca de 17-21 dias in vitro (DIV), as células microgliais estará pronto para a colheita a partir da cultura mista glial. Para determinar se as células estão prontos, examinar células sob um microscópio de contraste de fase invertida. Microglia estão prontos para a colheita quando a ~ 40% de confluência. Eles aparecem como células esféricas pequenas, brilhantes, crescendo como uma monocamada em cima de outro glia. </li></ol> 5. Isolamento da microglia de cultura mista de glia <ol><li> Para isolar microglia, vigorosamente tocar frasco contra a bancada de laboratório. Esta agitação ajuda microglia separadas de outras células da glia, devido às propriedades de baixa aderência da microglia. </li><li&gt; Selo T-75 tampas de garrafa com Parafilm e girar as culturas gliais mistos (200 rpm; 37 ° C, 5 h). usando um agitador rotativo nonhumidified com temperatura controlada Não permitir que as células a tremer por mais de 5 horas Inspecione visualmente. que microglia ter levantado da superfície do frasco depois de 5 horas, usando um microscópio de contraste de fase invertida. Microglia são luminosos, esférico, de livre flutuação células. Haverá uma camada residual de astrócitos e oligodendrócitos que aderiram ao fundo do frasco. </li><li> Recolhe meios de sobrenadante contendo microglia em um tubo de centrífuga estéril cónica; microglia pelota (215 xg, 5 min; rotor basculante). </li><li> Ressuspender o sedimento em microglia quente Microglial Meio de Crescimento (MGM; MEM suplementado com: 1 mM de piruvato de sódio, 0,6% v / vglucose, 1 mM de L-glutamina, 100 ug / ml de P / S, 5% v / v de FBS, 2 ~ ml / frasco), determinar a concentração de células utilizando um hemocitómetro, e a placa de 1 x 10 5 células / cavidade em um formato de placa de 24 poços emplástico ou deslizamentos estéreis tampa de vidro (500 mL / poço; MGM). Usando este procedimento, o rendimento médio microglia é de 7,5 x 10 5 células / filhote córtex. </li><li> 24 horas pós-chapeamento, remova todos os meios de comunicação e as células refeed com MGM fresco (37 ° C) para remover as células flutuantes e detritos. Esta etapa é fundamental para a sobrevivência da célula e manter microglia quiescentes. As células podem ser utilizados para a experimentação imediatamente após a realimentação. </li></ol> 6. Tratamentos Exemplo <ol><li> Expor as células a 10 ng / ml de Pam 3 CSK 4 (Pam), 10 ng / mL de lipopolissacarídeo (LPS) ou de controlo de veículo; incubar durante 2 ou 24 horas, numa incubadora humidificada (37 ° C, 5% CO 2). </li></ol> 7. Analisando Microglia para Funcionalidade Ex vivo via Immunofluorescent Imagem e Imunocitoquímica <ol><li> Após o tratamento, meios de cultura de células da colheita em tubos de microcentrífuga (1,5 ml) e centrifuge usando uma microcentrífuga de mesa (2 min; 900 xg) para limpar a mídia. Transferir o sobrenadante para um tubo de microcentrífuga limpo; ensaio imediatamente, ou armazenar a -20 ° C até que esteja pronto para uso. Analisar a concentração de TNF-α em células de cultura sobrenadante através disponível comercialmente kit de TNF-α rato ELISA. </li><li> Lavar as células com tampão fosfato salino (PBS), e fixar as células com 4% (w / v) de paraformaldeído / 4% (w / v) de sacarose / PBS pH 7,4 (4% PFA, 15 min; RT). Após a fixação, lavar as células com PBS durante 5 min com agitação suave num agitador orbital. <ol><li> Imunofluorescência / Imunocitoquímica. Após a fixação e PBS lavar, processo microglia para imunocitoquímica. Todas as etapas realizadas com agitação suave. Permeabilizar as células em PBS/0.1% de triton X-100, células de bloco em PBS/10% de soro de cabra normal durante uma hora. Incubar as células S / N a 4 ° C com anticorpo de coelho anti-NF-kB (p65 subunidade, 1/1, 250) ou de coelho anti-Iba-1 do anticorpo (1/750) no tampão de bloqueio. Visualize antigénio: anticorpo a seguir à incubação com fluorescência de cabra anti-coelho conjugado anticorpo IgG secundário (1 hora, 1/1, 000). Remover o anticorpo secundário não ligado por lavagem com PBS/0.1% de triton X-100 (3 x 5 min). </li><li> Células contra mancha com 4 &#39;,6-diamidino-2-fenilindole / PBS (DAPI, 0,1 ug / ml). De visualizar o núcleo da célula Todos os derivados de microglia CX3CR1-GFP + / - ratos expressam GFP. Utilizar um filtro de comprimento de onda de 350 nm para visualizar DAPI; de um filtro de comprimento de onda de 488 nm para visualizar GFP, e um filtro de comprimento de onda de 594 nm para visualizar p65 e Iba-1 imunocomplexos. </li></ol></li><li> Monte lamínulas utilizando solução aquosa de montagem e visualizar microglia por microscopia de fluorescência. </li></ol>

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	<li>Ranshoff, R. M. P. V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+Physiology:+Unique+Stimuli,+Specialized+Responses.">Microglia Physiology: Unique Stimuli, Specialized Responses.</a> Annu. Rev. Immunol. 27, 119-145 (2009).</li><li>Lawson, L. J., Perry, V. H., Dri, P., 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=Heterogeneity+in+the+distribution+and+morphology+of+microglia+in+the+normal+adult+mouse+brain.">Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain.</a> Neuroscience. 39, 151-170 (1990).</li><li>Gomez Perdiguero, E., Schulz, C., Geissmann, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+homeostasis+of+&amp;#34;resident&amp;#34;+myeloid+cells:+The+case+of+the+microglia.">Development and homeostasis of &amp;#34;resident&amp;#34; myeloid cells: The case of the microglia.</a> Glia. 61, 112-120 (2013).</li><li>Kierdorf, K., 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=Microglia+emerge+from+erythromyeloid+precursors+via+Pu.1-+and+Irf8-dependent+pathways.">Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways.</a> Nat. Neurosci. 16, 273-280 (2013).</li><li>Saijo, K., Glass, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cell+origin+and+phenotypes+in+health+and+disease.+Nature+reviews.">Microglial cell origin and phenotypes in health and disease. Nature reviews.</a> Immunology. , 11-775 (2011).</li><li>Davalos, D., 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=ATP+mediates+rapid+microglial+response+to+local+brain+injury+in+vivo.">ATP mediates rapid microglial response to local brain injury in vivo.</a> Nat. Neurosci. 8, 752-758 (2005).</li><li>Nimmerjahn, A., Kirchhoff, F., Helmchen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+microglial+cells+are+highly+dynamic+surveillants+of+brain+parenchyma+in+vivo.">Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo.</a> Science. 308, 1314-1318 (2005).</li><li>Hu, S., 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=Cytokine+and+free+radical+production+by+porcine+microglia.">Cytokine and free radical production by porcine microglia.</a> Clin. Immunol. Immunopathol. 78, 93-96 (1996).</li><li>Muzio, M., Polentarutti, N., Bosisio, D., Prahladan, M. K., Mantovani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptors:+a+growing+family+of+immune+receptors+that+are+differentially+expressed+and+regulated+by+different+leukocytes.">Toll-like receptors: a growing family of immune receptors that are differentially expressed and regulated by different leukocytes.</a> J. Leukocyte Biol. 67, 450-456 (2000).</li><li>Lee, S. J., Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptors+and+inflammation+in+the+CNS.">Toll-like receptors and inflammation in the CNS.</a> Curr. Drug Targets. Inflamm. Allergy. 1, 181-191 (2002).</li><li>Block, M. L., Zecca, L., Hong, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia-mediated+neurotoxicity:+uncovering+the+molecular+mechanisms.">Microglia-mediated neurotoxicity: uncovering the molecular mechanisms.</a> Nat. Rev. Neurosci. 8, 57-69 (2007).</li><li>Halle, A., 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=The+NALP3+inflammasome+is+involved+in+the+innate+immune+response+to+amyloid-beta.">The NALP3 inflammasome is involved in the innate immune response to amyloid-beta.</a> Nat. Immunol. 9, 857-865 (2008).</li><li>Chen, G. Y., Nunez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterile+inflammation:+sensing+and+reacting+to+damage.">Sterile inflammation: sensing and reacting to damage.</a> Nat. Rev. Immunol. 10, 826-837 (2010).</li><li>Duewell, P., 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=NLRP3+inflammasomes+are+required+for+atherogenesis+and+activated+by+cholesterol+crystals.">NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals.</a> Nature. 464, 1357-1361 (2010).</li><li>Stewart, C. R., 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=CD36+ligands+promote+sterile+inflammation+through+assembly+of+a+Toll-like+receptor+4.">CD36 ligands promote sterile inflammation through assembly of a Toll-like receptor 4.</a> 11, 155-161 (2010).</li><li>Beraud, D., 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=alpha-Synuclein+Alters+Toll-Like+Receptor+Expression.">alpha-Synuclein Alters Toll-Like Receptor Expression.</a> Front. Neurosci. 5, 80 (2011).</li><li>Banati, R. B., Gehrmann, J., Schubert, P., Kreutzberg, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxicity+of+microglia..">Cytotoxicity of microglia..</a> Glia. 7, 111-118 (1993).</li><li>Combs, C. K., Karlo, J. C., Kao, S. C., Landreth, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Amyloid+stimulation+of+microglia+and+monocytes+results+in+TNF+alpha-dependent+expression+of+induciblenitric+oxide+synthase+and+neuronal+apoptosis.">beta-Amyloid stimulation of microglia and monocytes results in TNF alpha-dependent expression of induciblenitric oxide synthase and neuronal apoptosis.</a> J. Neurosci. 21, 1179-1188 (2001).</li><li>Kim, Y. S., Joh, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia,+major+player+in+the+brain+inflammation:+their+roles+in+the+pathogenesis+of+Parkinson's+disease.">Microglia, major player in the brain inflammation: their roles in the pathogenesis of Parkinson's disease.</a> Exp. Mol. Med. 38, 333-347 (2006).</li><li>Colton, C., Wilcock, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+activation+states+in+microglia.">Assessing activation states in microglia.</a> CNS Neurol. Disord. Drug Targets. 9, 174-191 (2010).</li><li>Nakajima, K., Tohyama, Y., Kohsaka, S., Kurihara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ceramide+activates+microglia+to+enhance+the+production/secretion+of+brain-derived+neurotrophic+factor+(BDNF)+without+induction+of+deleterious+factors+in+vitro.">Ceramide activates microglia to enhance the production/secretion of brain-derived neurotrophic factor (BDNF) without induction of deleterious factors in vitro.</a> J. Neurochem. 80, 697-705 (2002).</li><li>Uesugi, M., Nakajima, K., Tohyama, Y., Kohsaka, S., Kurihara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonparticipation+of+nuclear+factor+kappa+B+(NFkappaB)+in+the+signaling+cascade+of+c-JunN-terminal+kinase+(JNK)-+and+p38+mitogen-activated+protein+kinase+(p38MAPK)-dependent+tumor+necrosis+factor+alpha+(TNFalpha)+induction+in+lipopolysaccharide+(LPS)-stimulated+microglia.">Nonparticipation of nuclear factor kappa B (NFkappaB) in the signaling cascade of c-JunN-terminal kinase (JNK)- and p38 mitogen-activated protein kinase (p38MAPK)-dependent tumor necrosis factor alpha (TNFalpha) induction in lipopolysaccharide (LPS)-stimulated microglia.</a> Brain Res. , 1073-1074 (2006).</li><li>Beraud, D., 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=Microglial+Activation+and+Antioxidant+Responses+Induced+by+the+Parkinson's+Disease+Protein+alpha-Synuclein.">Microglial Activation and Antioxidant Responses Induced by the Parkinson's Disease Protein alpha-Synuclein.</a> J. Neuroimmun. Pharmacol. 8, 94-117 (2013).</li><li>Su, X., 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=Synuclein+activates+microglia+in+a+model+of+Parkinson's+disease.">Synuclein activates microglia in a model of Parkinson's disease.</a> Neurobiol. Aging. 29, 1690-1701 (2008).</li><li>Minghetti, L., Levi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+as+effector+cells+in+brain+damage+and+repair:+focus+on+prostanoids+and+nitric+oxide.">Microglia as effector cells in brain damage and repair: focus on prostanoids and nitric oxide.</a> Prog. Neurobiol. 54, 99-125 (1998).</li><li>Hirsch, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cells+and+Parkinson's+disease.">Glial cells and Parkinson's disease.</a> J. Neurol.. 247 (2), 58-62 (2000).</li><li>Liu, B., 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=Role+of+nitric+oxide+in+inflammation-mediated+neurodegeneration.">Role of nitric oxide in inflammation-mediated neurodegeneration.</a> Ann. NY Acad. Sci. 962, 318-331 (2002).</li><li>Shie, F. S., Nivison, M., Hsu, P. C., Montine, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+microglialinnate+immunity+in+Alzheimer's+disease+by+activation+of+peroxisome+proliferator-activated+receptor+gamma.">Modulation of microglialinnate immunity in Alzheimer's disease by activation of peroxisome proliferator-activated receptor gamma.</a> Curr. Med. Chem. 16, 643-651 (2009).</li><li>Rogers, J., Mastroeni, D., Leonard, B., Joyce, J., Grover, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinflammation+in+Alzheimer's+disease+and+Parkinson's+disease:+are+microglia+pathogenic+in+either+disorder.">Neuroinflammation in Alzheimer's disease and Parkinson's disease: are microglia pathogenic in either disorder.</a> Int. Rev. Neurobiol. 82, 235-246 (2007).</li><li>Cardona, A. E., Huang, D., Sasse, M. E., Ransohoff, R. M. <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+murinemicroglial+cells+for+RNA+analysis+or+flow+cytometry.">Isolation of murinemicroglial cells for RNA analysis or flow cytometry.</a> Nat. Protoc. 1, 1947-1951 (2006).</li><li>Ford, A. L., Goodsall, A. L., Hickey, W. F., Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+adult+ramified+microglia+separated+from+other+central+nervous+system+macrophages+by+flow+cytometric+sorting.+Phenotypic+differences+defined+and+direct+ex+vivo+antigenpresentation+to+myelin+basic+protein-reactive+CD4++T+cells+compared.">Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigenpresentation to myelin basic protein-reactive CD4+ T cells compared.</a> J. Immunol. 154, 4309-4321 (1995).</li><li>Ford, A. L., Foulcher, E., Goodsall, A. L., Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+digestion+with+dispase+substantially+reduces+lymphocyte+and+macrophage+cell-surface+antigen+expression.">Tissue digestion with dispase substantially reduces lymphocyte and macrophage cell-surface antigen expression.</a> J. Immunol. Methods. 194, 71-75 (1996).</li><li>Pino, P. A., Cardona, A. E. <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+brain+and+spinal+cord+mononuclear+cells+using+percoll+gradients.">Isolation of brain and spinal cord mononuclear cells using percoll gradients.</a> J. Vis. Exp. , (2011).</li><li>Veremeyko, T., Starossom, S. C., Weiner, H. L., Ponomarev, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+microRNAs+in+microglia+by+real-time+PCR+in+normal+CNS+and+during+neuroinflammation.">Detection of microRNAs in microglia by real-time PCR in normal CNS and during neuroinflammation.</a> J. Vis. Exp. , (2012).</li><li>Su, X., Federoff, H. J., Maguire-Zeiss, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+alpha-synuclein+overexpression+mediates+early+proinflammatory+activity.">Mutant alpha-synuclein overexpression mediates early proinflammatory activity.</a> Neurotox. Res. 16, 238-254 (2009).</li><li>Jung, S., 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=Analysis+of+fractalkine+receptorCX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptorCX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol. Cell. Biol. 20, 4106-4114 (2000).</li><li>Imai, T., 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=Identification+and+molecular+characterization+of+fractalkine+receptor+CX3CR1,+which+mediates+both+leukocyte+migration+and+adhesion.">Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion.</a> Cell. 91, 521-530 (1997).</li><li>Cardona, A. E., 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=Control+of+microglial+neurotoxicity+by+the+fractalkine+receptor.">Control of microglial neurotoxicity by the fractalkine receptor.</a> Nat. Neurosci. 9, 917-924 (2006).</li><li>Streit, W. J., Walter, S. A., Pennell, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+microgliosis.">Reactive microgliosis.</a> Prog. Neurobiol. 57, 563-581 (1999).</li><li>Frank, M. G., Wieseler-Frank, J. L., Watkins, L. R., Maier, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+highly+enriched+and+quiescent+microglia+from+adult+rat+hippocampus:immunophenotypic+and+functional+characteristics.">Rapid isolation of highly enriched and quiescent microglia from adult rat hippocampus:immunophenotypic and functional characteristics.</a> J. Neurosci. Methods. 151, 121-130 (2006).</li></ol>

Os autores declaram que a pesquisa foi conduzida na ausência de quaisquer relações comerciais ou financeiras que possam ser interpretadas como um potencial conflito de interesses.

O presente processo oferece um método eficaz para o isolamento da microglia corticais de ratinhos neonatais. Este procedimento tem um benefício duplo de 1) a preservação imunofenotipagem microglia e funcionalidade, como determinado por imagiologia fluorescente, imunocitoquímica, e ELISA, e, 2), permitindo que a microglia para amadurecer na presença de outras células gliais (astrócitos e oligodentrocytes) antes isolamento, o que pode facilitar as interações célula-célula importantes durante o período de matu...

Células microgliais, os macrófagos de vigilância do parênquima do SNC, compreendem cerca de 12% da população total de células de cérebro de mamíferos adultos. Microglia são derivadas do saco vitelino mielóides células precursoras que variam em densidade celular e morfologia em diferentes regiões citoarquitecturais dentro do SNC adulto 1-5. Em um adulto saudável do cérebro, microglia são células pequenas, ramificadas ou polares, com processos dinâmicos finas. Em contraste com a morfologia dos macrófagos periférica, microglia demonstrar um repouso, fenótipo de baixo perfil em cérebros saudáveis ​​que podem aparecer como inatividade celular, no entanto, estudos in vivo de imagens demonstram que processos microgliais estender dinamicamente e retrair para monitorar seu microambiente de uma forma que lembra &quot; amostragem e levantamento &quot;6,7. Microglia são altamente responsivos e diferencialmente a alterações ambientais e fisiopatológicos no cérebro, a mudançaa partir de um vigilante de um estados considerados como seu repouso e activado comumente no estado efectoras, respectivamente. Esta chave na ativação pode ser mediada por um envolvimento de receptores ligados à membrana de reconhecimento de padrões (PRRs), tais como receptores toll-like (TLRs), que respondem a padrões moleculares associados a patógenos (PAMPs), lipoproteínas nomeadamente bacterianas e virais derivadas , ácidos nucleicos, hidratos de carbono e 8-11. Além de PAMPs, PRRs também foram mostrados para induzir a ativação da microglia contra moléculas estéreis, não patogênicas, conhecidos como padrões moleculares perigo / associado a danos (amortece), que representam uma perturbação na homeostase do SNC, tais como danos celulares 12-16. Uma vez envolvido, PRRs iniciar uma cascata de sinalização intracelular, que resulta em alterações na morfologia celular e a expressão do gene da microglia, mais especificamente, a microglia activada adaptar um fenótipo amebóides semelhante, translocar o p65 de NF-kB subunidade (p65) para o núcleo da célula, e regula positivamente a producção e secreção de citocinas pró-inflamatórias, tais como fator de necrose tumoral-α (TNF-α), interleucina-1 β (IL-1β), juntamente com espécies reativas de oxigênio (ROS) 16-24. Embora integrante da resposta imune inata do sistema nervoso central, estas moléculas segregadas também foram encontradas para aumentar o stress oxidativo neuronal, induzindo e exacerbar a neurodegeneração em estados patológicos, tais como a doença de Parkinson e doença de Alzheimer 25-29. No entanto, os mecanismos de ativação da microglia em estados patológicos não estão completamente esclarecidos. Portanto, o isolamento de microglia é uma poderosa ferramenta de investigação para esses processos biológicos, como muitos em características in vivo de ativação da microglia pode ser reproduzido na cultura. Vários métodos estão disponíveis para o isolamento de microglia, incluindo o isolamento por meio de um gradiente de Percoll a seguir à digestão enzimática de tecido do SNC 30,31. No entanto, a digestão enzimática pode alterara imunofenotipagem de células por redução da superfície da célula expressão de antigénios de 32, e resulta na produção de células por animal menor do que o método aqui descrito. Especificamente, nós relatamos um rendimento médio por microglia filhote córtex de 7,5 x 10 5 células, enquanto anteriormente relatados métodos de isolamento de todo CNS através de um gradiente de Percoll rendimento 3-5 x 10 5 células 30,33,34. O presente processo evita a utilização de enzimas de digestão, isolando microglia com base nas suas propriedades de baixa aderência, preservando assim a imunofenotipagem da microglia e funcionalidade. No presente estudo, descrevemos o isolamento de células microgliais de culturas mistas gliais derivadas de heterozigotos CX3CR1-GFP (CX3CR1-GFP + / -) neonatal e C57BL / 6 córtices murino via agitação mecânica num agitador rotativo, uma extensão do anteriormente método publicado 24,35. Nós utilizamos o ex-tensão do mouse para fácil visualização da microglia, comoestes ratinhos expressam GFP sob o controlo do locus endógeno CX3CR1 - um promotor específico de monócitos 36-38. Este método produz culturas microgliais altamente puros com um imunofenotipagem conservados ex vivo, como demonstrado por alterações morfológicas, a translocação nuclear de p65, e a secreção de TNF-α quando desafiados com o lipopolissacarídeo bacteriano (LPS) ou Pam 3 CSK 4, TLR4 e TLR1 / 2 agonistas , respectivamente.

<table border="0" cellpadding="0" cellspacing="0" width="680">
	<colgroup>
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	</colgroup>
	<tbody>
		<tr height="160">
			<td height="160" style="height:160px;width:233px;">Glucose</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:155px;">G8270</td>
			<td style="width:143px;">Make 20% Stock Solution with MilliQ water; filter sterilize; store at 4 &#176;C; shelf life: 3-6 months. Used to make MCM and MGM.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Sodium pyruvate 100 mM (100x)</td>
			<td style="width:149px;">Hyclone</td>
			<td style="width:155px;">SH30239.01</td>
			<td style="width:143px;">Store at 4 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Penicillin/Streptomycin 10,000 units/ml (100x)</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">15140-122</td>
			<td style="width:143px;">Store at -20 &#176;C. Used to make MCM, MGM, MM, and DM.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">L-glutamine 200 mM (100x)</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">25030-081</td>
			<td style="width:143px;">Store at -20 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Fetal Bovine Serum (Defined)</td>
			<td style="width:149px;">Hyclone</td>
			<td style="width:155px;">SH30070.03</td>
			<td style="width:143px;">Filter sterilize; store at -20 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:233px;">Minimum Essential Medium Earle&#39;s (MEM)</td>
			<td style="width:149px;">Cellgro</td>
			<td style="width:155px;">15-010-CV</td>
			<td style="width:143px;">Without L-glutamine. Contains Earle&#39;s salts. Used to make MCM, MGM, and DM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Horse Serum</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">16050</td>
			<td style="width:143px;">Filter sterilize; store at -20 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td style="width:149px;">Cellgro</td>
			<td style="width:155px;">21-021-CV</td>
			<td style="width:143px;">Without calcium and magnesium. Store at 4 &#176;C. Used to make MM.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">HEPES 1 M</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">15630-031</td>
			<td style="width:143px;">Store at 4 &#176;C. Used to make MM.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">T-75 Flask</td>
			<td style="width:149px;">Corning</td>
			<td style="width:155px;">430641</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">4&#39;,6-Diamidino-2-Phenylindole, dilactate (DAPI)</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:155px;">D3571</td>
			<td style="width:143px;">Used to stain cell nucleus.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Rabbit anti-Iba1</td>
			<td style="width:149px;">Wako</td>
			<td style="width:155px;">019-19741</td>
			<td style="width:143px;">Used at 1/750 dilution for ICC staining of Iba1.&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Rabbit anti-NF&#954;B (p65)</td>
			<td style="width:149px;">Abcam</td>
			<td style="width:155px;">7970</td>
			<td style="width:143px;">Used at 1/1250 dilution for ICC staining for p65.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:233px;">Alexafluor 594 Goat anti-Rabbit IgG (H+L)</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:155px;">A11012</td>
			<td style="width:143px;">Used at 1/1000 dilution for visualization of antigen:antibody complex in ICC.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">10 ml Disposable Serological Pipet</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">13-678-11E</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">50 ml Disposable Centrifuge Tube</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">05-539-8</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">15 ml Disposible Centrifuge Tube</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">05-539-12</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Sterile Polystyrene Petri Dish</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">875713</td>
			<td style="width:143px;">100 mm x 15 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Scissor: Straight Metzembaum (scissor #1)</td>
			<td style="width:149px;">Roboz Surgical</td>
			<td style="width:155px;">RS-6010</td>
			<td style="width:143px;">1; 5 inch; used for removing head</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Scissor: Vannas 
			(scissor #2)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">15000-08</td>
			<td style="width:143px;">1; non-angled; 2.5mm cutting edge; used to open scalp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Scissor: Student Vannas (scissor #3)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">91501-09</td>
			<td style="width:143px;">1; curved; used to mince brain tissue</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Forcep: Dumont #7 
			(forcep #1)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">91197-00</td>
			<td style="width:143px;">2; used to secure nose and remove cortices&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Forcep: Dumont #2 
			(forcep #2)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">11223-20</td>
			<td style="width:143px;">1; used to remove scalp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Forcep: Dumont #3 
			(forcep #3)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">11231-30</td>
			<td style="width:143px;">1; used to remove skull</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Forcep: Dumont #5a 
			(forcep #4)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">11253-21</td>
			<td style="width:143px;">1; used to remove meninges</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Table of specific equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Name of Equipment</td>
			<td style="width:149px;">Name of Company</td>
			<td style="width:155px;">Catalogue Number</td>
			<td style="width:143px;">Comments</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Zoom Stereo Dissection Microscope&#160;</td>
			<td style="width:149px;">Olympus</td>
			<td style="width:155px;">SZ4060</td>
			<td style="width:143px;">Microscope is placed inside Laminar-Horizontal Flow Cabinet</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Laminar-Horizontal Flow Cabinet</td>
			<td style="width:149px;">Nuaire</td>
			<td style="width:155px;">NU-201-330</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Biological Safety Cabinet</td>
			<td style="width:149px;">Labconco</td>
			<td style="width:155px;">3440001</td>
			<td style="width:143px;">Class II</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:233px;">Water-Jacketed CO2 Incubator</td>
			<td style="width:149px;">VWR</td>
			<td>97025-836</td>
			<td style="width:143px;">Set to 37 &#176;C, 5% CO2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Swing-out buckets</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>75006441</td>
			<td style="width:143px;">To be used with Swing-out rotor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Swing-out Rotor</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>75006445</td>
			<td style="width:143px;">Max Radius: 19.2 (cm)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Sorvall Legend RT+ Centrifuge 
			(clinical centrifuge)</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>75-004-377</td>
			<td style="width:143px;">With swing-out rotor</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">AccuSpin Micro 17 microcentrifuge 
			(tabletop microcentrifuge)</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>S98645</td>
			<td style="width:143px;">With microliter rotor (24 x 1.5/2.0 ml; 
			Cat #: 75003524)&#160;&#160;</td>
		</tr>
	</tbody>
</table>

isolation of cortical microglia with preserved immunophenotype and functionality from murine neonates

Isolamento de microglia de tecido do SNC é um poderoso instrumento de investigação utilizado para estudar biologia microglial ex vivo. O presente método de detalhes de um procedimento para o isolamento da microglia de córtices murino neonatal por agitação mecânica com um agitador rotativo. Esse isolamento microglia método produz microglia corticais altamente puras que apresentam características morfológicas e funcionais indicativos de microglia quiescentes em condições não patológicas normais in vivo. Este procedimento também preserva a imunofenotipagem da microglia e funcionalidade bioquímica como demonstrado pela indução de alterações morfológicas, translocação nuclear da subunidade p65 de NF-kB (p65), e a secreção de citoquina pró-inflamatória característica, fator de necrose tumoral-α (TNF-α ), ao lipopolissacarídeo (LPS) e Pam 3 CSK 4 (Pam) desafios. Portanto, o processo de isolamento do presente preserva a imunofenotipagem de ambos quiescente e actimicroglia motivadas, proporcionando um método experimental de investigação de biologia microglia em condições ex vivo.

Mantendo a imunofenotipagem e funcionalidade da microglia ex vivo durante o isolamento é importante ser capaz de utilizar essas células como um modelo de investigação para a biologia da microglia. A fim de demonstrar a conservação bem sucedida da microglia immunofunctionality usando o presente método, foram isoladas a partir de recém-nascidos microglia corticais P3 (CX3CR1-GFP + / - C57BL / 6) e as culturas tratadas quer com LPS ou Pam. Como ilustrado na F...

Watch this Scientific Journal Video about Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates at JoVE.com

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

Uma chave para o sucesso na investigação de biologia da microglia é a preservação do immunofunction microglial ex vivo durante o isolamento a partir de tecido do SNC. Isolando microglia através de resultados de agitação rotativos em culturas de células altamente puras e imuno avaliado pela imagem fluorescente, imunocitoquímica e ELISA após a ativação da microglia com o lipopolissacarídeo estímulos pró-inflamatória (LPS) e Pam 3 CSK 4 (Pam).

Isolamento de Cortical Microglia com Imunofenótipo preservada e funcionalidades de murino Os recém-nascidos

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

isolation-cortical-microglia-with-preserved-immunophenotype

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JoVE Journal

Immunology and Infection

15.7K visualizações.  Georgetown University Medical Center.  Uma chave para o sucesso na investigação de biologia da microglia é a preservação do immunofunction microglial ex vivo durante o isolamento a partir de tecido do SNC. Isolando microglia através de resultados de agitação rotativos em culturas de células altamente puras e imuno avaliado pela imagem fluorescente, imunocitoquímica e ELISA após a ativação da microglia com o lipopolissacarídeo estímulos pró-inflamatória (LPS) e Pam 3 CSK 

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

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.

Isolamento Microglia primária a partir de cultura mista de células gliais de tecido de cérebro de rato neonatal

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

Imunologia e Infecção

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

Isolamento, processamento e análise de células gengivais murino

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

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

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

Microglia cultura do Sistema Nervoso Central Neonatal e Adulto

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

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Isolamento, identificação e purificação de células epiteliais do timo murino

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Isolamento de Murino Linfonodo células estromais

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

Isolamento e Transplante de diferentes envelhecidas murino tímicos enxertos.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

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

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

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

Isolamento de Leucócitos a partir de tecidos de murino na interface materno-fetal

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

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Isolamento e Activação de Linfócitos murinos

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Análise de Linfócitos extravasamento Usando um<em&gt; In Vitro</em&gt; Modelo do Sangue-Cérebro humano Barreira

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

Isolamento de Cortical Microglia com Imunofenótipo preservada e funcionalidades de murino Os recém-nascidos

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