Este trabalho foi financiado por uma bolsa de investigação e um Grant-in-Aid do Projeto de Alimentos Nanotecnologia do Ministério da Agricultura, Florestas e Pesca do Japão.

1. Perfusão do Fígado <ol><li> Anestesiar um adulto de ratos Sprague-Dawley do sexo masculino (de 10 a 15 semanas de idade; peso corporal 150-200 g) por uma injeção intraperitoneal de uma solução de pentobarbital sódico (50 mg de sódio pentobarbital / kg de peso corporal). </li><li> Colocar o animal em uma panela inoxidável e spray de etanol a 70% em seu abdômen e tórax. </li><li> Faça um pequeno corte e retire a pele. Abrir o abdômen pelo corte com uma tesoura. </li><li> Confirmar a localização da veia porta, passe um fio cirúrgico sob a veia porta e vagamente moita. </li><li> Moita a parte distal da veia cava inferior e veia mesentérica para evitar o refluxo de sangue para o fígado. Abrir a cavidade torácica, cortando o diafragma com uma tesoura, e expor o coração. </li><li> Inserir um cateter plástico (2 mm de diâmetro) na veia porta e apertar firmemente o fio ao redor do cateter. </li><li> Conectar o cateter a um sistema de perfusão que é carregado com a solução de lavagem (Ca 2 +-Mg 2 + livre HBSS contendo 0,5 mM EGTA, 10 HEPES mM e 4,2 mM NaHCO 3, pH 7,2) pré-aquecido a 37 ° C. </li><li> Comece a perfusão in situ a uma taxa de 10 ml / min. Ao mesmo tempo, faça um corte na parede do átrio direito. Continue perfusão por 10 min. </li><li> Mudar a solução de perfusão para a solução de colagenase [HBSS contendo HEPES 10 mM e NaHCO 3 4,2 suplementado com colagenase tipo I (0,05%) e inibidor de tripsina (50 mcg / ml), pH 7.5], perfundir e por 10 - 20 min a uma taxa de 10 ml / min, até o fígado incha um pouco e mostra o sinal para a desintegração da estrutura do fígado sob membrana serosa. </li></ol> 2. Preparação de Fração celular no parênquima Hepatócito ricos <ol><li> Remover e transferir cuidadosamente o fígado, para um copo estéril contendo 25 ml de solução de colagenase. Picar em pedaços pequenos com tesoura. </li><li> Adicionar 75 ml de MEM frio para a suspensão celular resultante. Após pipetar gentil, filtrado através de um filtro de células (100 mm) para remover o tecido conjuntivo e fragmentos de tecido não digerido. </li><li> Transfira o filtrado para 50 ml tubos cônicos e centrifugar a 50 xg por 1 min a 4 ° C (freio). </li><li> Cuidadosamente desprezar o sobrenadante. Ressuspender o sedimento com MEM frio. </li><li> Repita 2.4) três vezes. </li></ol> 3. Cultura primária mista de células do fígado <ol><li> Suspender as células obtidas em 2.5) no meio de crescimento composto por DMEM contendo 10% de calor FCS inativada, suplementado com 100 mM β-mercaptoetanol, 10 de insulina mcg / ml, estreptomicina 100 mg / ml e 100 U / ml de penicilina, e sementes em 09:55 frascos de cultura de tecido (superfície: 75 cm 2) com uma densidade de 6,7 x 10 4 células / cm 2 (5 x 10 6 células / frasco / 12 -15 ml de meio). </li><li> Incubar a cultura flasksat 37 ° C em uma atmosfera de 5% CO 2-95% de ar. </li><li> Substituir o meio de crescimento a cada 2 a 3 intervalos de dias. </li></ol> 4. Isolamento seletivo de macrófagos <ol><li> Após 12 dias de cultura, os macrófagos proliferam na folha de celular. Estas células são suspensas por recíproca agitação dos frascos de cultura em 80-120 golpes por minuto por 30 min a 37 ° C. </li><li> Transferência do meio de cultura em 100 milímetros de tecido não-cultura grade pratos de plástico. Piscina meio de dois frascos de T75 em um prato de plástico. Frascos de cultura de recarga com o meio de crescimento e devolvê-los à incubadora. </li><li> Incubar pratos de plástico por 30 min a 37 ° C em uma atmosfera de 5% de CO 2-95% de ar. Macrófagos facilmente anexar ao tecido não-cultura pratos grade de plástico no âmbito do presente processo de incubação, enquanto que outras células não contaminar. </li><li> Aspirar a médio e lave o prato de plástico com PBS. Repita este passo três vezes. </li><li> Adicionar 1 ml de solução TrypLE Express para o prato, e incubar por 10 min a 37 ° C em uma atmosfera de 5% de CO 2-95% de ar. </li><li> Adicionar 9 ml do meio de crescimento. Delicadamente raspar os macrófagos presa por um raspador de celular. </li><li> Transferir a suspensão de células em um tubo cônico de 15 ml e centrifugar a 180 xg por 5 min a 25 ° C (freio). </li><li> Desprezar o sobrenadante e adicionar 1 ml do meio de crescimento. Dissociar-se em células isoladas por pipetagem vigoroso. Enumerar o número de células por um hemocitômetro contagem. </li><li> Medidas de isolamento pode ser repetido com a mesma cultura em frascos de 2-3 intervalos de dia para mais de duas semanas. </li><li> Macrófagos isolados podem ser cultivadas em meio de cultura descrito no ponto 3.1. </li></ol> 5. Resultados representante Um exemplo de uma cultura mista primária de células do fígado de ratos adultos é mostrado na Figura 2. Hepatócitos do parênquima perder a morfologia das células epiteliais dentro de poucos dias em cultura, degenerados ou transformar em fibroblasto-como pilhas.Então, por volta do dia 6 a 12, de contraste de fase brilhante, redondo macrófagos como células vigourouly proliferam na camada de células fibroblásticas. Por agitação dos frascos de cultura, os macrófagos são facilmente suspensos no meio de cultura, e sua posterior transferência e incubação em pratos de plástico resultam em adesão seletiva de macrófagos (Figura 3). As células macrófagos-like podem ser colhidas mais cedo dia 8, e os números atingiram níveis máximos nos dias 12 a 14. Mais de 10 6 células podem ser colhidas a partir de uma garrafa de cultura T75 repetidamente em intervalos de 2-3 dias por mais de duas semanas, permitindo um rendimento total de células por frasco de 10 7 por T75 frasco de cultura (Figura 3). A pureza dos macrófagos isolados foram de 95 a 99%, avaliada pelo flowcytometry ou imunocitoquímica com ratos macrófagos anticorpos específicos, como o ED-1 (Figura 4), ​​ED-3, OX-41 (Figura 4) e Iba1 9. As células isoladas mostram características típicas de macrófagos, tais como fagocitose ativa de contas polystylene (Figura 5), a resposta proliferativa a GM-CSF recombinante, a secreção de inflamatórias / citocinas anti-inflamatórias após estimulação com LPS, e formação de células gigantes multinucleadas 9. <img src="/files/ftp_upload/2757/2757fig1.jpg" alt="Figura 1" /> Figura 1. Um esquema de isolamento seletivo e purificação de macrófagos como células de cultura primária mista de células de fígado de rato. <img src="/files/ftp_upload/2757/2757fig2.jpg" alt="Figura 2" /> Figura 2. Cultura primária de células de fígado de rato e proliferação de macrófagos como células. Seta indica uma célula gigantes multinucleadas. Barra de escala = 100 mm. Reproduzido do Jornal de métodos imunológicos, Vol.360, 47-55 (2010) 9 com permissão da Elsevier. <img src="/files/ftp_upload/2757/2757fig3.jpg" alt="Figura 3" /> Figura 3. Isolamento selectivo de macrófagos como células pela agitação e método de fixação. As células foram suspensas em meio de cultura por agitação dos frascos, posteriormente transferida para o tecido não-cultura pratos de plástico de grau, e incubados a 37 ° C. Já em 10 min após o plaqueamento, macrófagos como células aderidas à superfície do prato, enquanto outros contaminar células fibroblásticas permaneceu suspenso. Após lavagem com PBS, uma população de macrófagos altamente purificada foi obtida. Estas células ganhou a morfologia típica de macrófagos após 40 min da cultura, e as células mitóticas foram freqüentemente observadas (setas). Alterações subsequentes no número de células recuperado de frascos em períodos diferentes culturas são mostradas. Os valores são em média ± SD 3-5 frascos. Experimentos foram repetidos três vezes e os dados são indicados por símbolos diferentes. Barra de escala = 100 mm. Reproduzido do Jornal de métodos imunológicos, Vol.360, 47-55 (2010) 9 com permissão da Elsevier. <img src="/files/ftp_upload/2757/2757fig4.jpg" alt="Figura 4" /> Figura 4. Coloração imunocitoquímico dos macrófagos como células com anticorpos monoclonais contra macrófagos de ratos. <img src="/files/ftp_upload/2757/2757fig5.jpg" alt="Figura 5" /> Figura 5. Fagocitose de FITC marcado por microesferas de macrófagos como células.

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

Todos os animais foram mantidos e operados na unidade animal no Instituto Nacional de Saúde Animal, de acordo com as diretrizes e regulamentos estabelecidos pelo Comitê da Instituição para experimentos com animais.

Aqui, nós relatamos um método simples e eficiente para obter macrófagos da mistura de culturas primárias de células do fígado de ratos adultos. Nosso método baseia-se primeiro sobre a atividade proliferativa romance de macrófagos na cultura mista de células de fígado de rato, e depois posterior isolamento e purificação dessas células, com base em suas características biológicas como macrófagos 9. Pode ser possível os macrófagos proliferaram na cultura mista primária de células do fígado adulto pode ter se originado a partir de Kupffer ou células relacionadas que contaminou a fração em hepatócitos do parênquima. Os macrófagos podem ter respondido a alterações específicas ambientais causados ​​pela transformação do parênquima hepatócito 10 e outras células fibroblásticas na mistura de culturas primárias. Em conclusão, nosso método proporciona isolamento dos macrófagos como células em número suficiente e pureza da cultura primária de fígado de rato cellswithout usando equipamentos sofisticados e habilidades técnicas. Além disso, o procedimento de isolamento pode ser repetido com o frasco mesma cultura por mais de duas semanas. Esse método pode ser aplicável a outras espécies de mamíferos.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Solução de pentobarbital sódico </td><td> Kyoritsu Seiyaku </td><td> Injeção Somnopentyl </td><td></td></tr><tr><td> Solução salina balanceada de Hank (HBSS) </td><td> Invitrogen </td><td> 14185-052 </td><td></td></tr><tr><td> Etilenoglicol tetraacetic ácido (EGTA) </td><td> Sigma-Aldrich </td><td> E-4378 </td><td></td></tr><tr><td> Colagenase </td><td> Wako Pure Chemical Industries, Ltd. </td><td> 032-10534 </td><td> Dissociação celular grade (Lot verifique necessário) </td></tr><tr><td> Inibidor de tripsina </td><td> Sigma-Aldrich </td><td> T-9128 </td><td></td></tr><tr><td> Meio mínimo essencial de Eagle (MEM) </td><td> Sigma-Aldrich </td><td> M-4655 </td><td></td></tr><tr><td> Dulbecco modificado Meio de Eagle (DMEM) </td><td> Sigma-Aldrich </td><td> D-6459 </td><td> Alta de glicose do tipo </td></tr><tr><td> Soro fetal bovino (FCS) </td><td> HyClone </td><td> SH30070.03 </td><td> Inativados pelo calor a 56 ° C por 30 min. (Lot verifique necessário) </td></tr><tr><td> TrypLE expresso </td><td> Invitrogen </td><td> 12605 </td><td></td></tr><tr><td> Fosfato de Dulbecco solução salina tamponada (PBS) </td><td> Invitrogen </td><td> 14190 </td><td> Ca 2 +-Mg 2 + livre </td></tr><tr><td> β-mercaptoetanol </td><td> Sigma-Aldrich </td><td> M-7522 </td><td> Stock: 100 mM em água destilada </td></tr><tr><td> Insulina </td><td> Sigma-Aldrich </td><td> I-5500 </td><td> Stock: 10 mg / ml em HCl 0,1 N </td></tr><tr><td> Penicilina / estreptomicina </td><td> Invitrogen </td><td> 15070 </td><td></td></tr><tr><td> Coador celular </td><td> BD Biosciences </td><td> 352360 </td><td> Malhagem: 100 mm </td></tr><tr><td> Frasco de cultura de tecidos </td><td> Sumitomo Bakelite Co., Ltd. </td><td> MS-21250 </td><td> Superfície: 75 cm 2 </td></tr><tr><td> Não-tecido placas de cultura de plástico </td><td> BD Biosciences </td><td> 351005 </td><td></td></tr><tr><td> Scraper celular </td><td> Corning Inc. </td><td> 3010 </td><td></td></tr><tr><td> Shaker recíproco </td><td> TAITEC </td><td> NR-1 </td><td></td></tr></tbody></table>

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

Células de Kupffer são macrófagos hepáticos específicos residente e desempenhar um papel importante nas funções fisiológicas e patológicas do fígado 1-3. Embora os métodos de isolamento de macrófagos do fígado têm sido bem descrita 4-6, a maioria destes métodos requerem equipamentos sofisticados, como um elutriator centrífuga e habilidades técnicas. Aqui, nós fornecemos um novo método para obter macrófagos de fígado em número suficiente e pureza da mistura culturas primárias de células do fígado de ratos adultos, como esquematicamente ilustrada na Figura 1. Após a dissociação das células do fígado por duas etapas método de perfusão 7,8, uma fração na sua maioria composta de hepatócitos do parênquima é preparado e semeado em cultura de tecidos T75 frascos com meio de cultura composto por DMEM e 10% hepatócitos FCS.Parenchymal perder a morfologia das células epiteliais dentro de poucos dias em cultura, degenerados ou transformar em fibroblasto-como as células (Figura 2). Como o produto da cultura, por volta do dia 6, de contraste de fase brilhante, redondo macrófagos-like células começam a proliferar na camada de células fibroblásticas (Figura 2). O crescimento das células macrófagos-like continuar e chegar ao nível máximo, em torno de 12 dias, cobrindo a camada de células na superfície do frasco. Por agitação dos frascos de cultura, os macrófagos são facilmente suspensos no meio de cultura. Posterior transferência e incubação curto, em resultado pratos de plástico na adesão seletiva de macrófagos (Figura 3), enquanto que outras células contaminantes permanecem suspensas. Depois de várias lavagens com PBS, os macrófagos são colhidas em anexo. Mais de 10 6 células podem ser colhidas várias vezes da mesma garrafa de cultura de tecidos T75 menos duas a três intervalos dias por mais de duas semanas (Figura 3). Purezas dos macrófagos isolados foram de 95 a 99%, avaliada por citometria de fluxo ou imunocitoquímica com ratos macrófagos anticorpos específicos (Figura 4). células isoladas mostram fagocitose ativa de contas polystylene (Figura 5), ​​a resposta proliferativa a GM-CSF recombinante, a secreção de inflamatórias / citocinas anti-inflamatórias após estimulação com LPS, e formação de células gigantes multinucleadas 9. Em conclusão, nós fornecemos um método simples e eficiente para obter macrófagos de fígado em número suficiente e pureza sem equipamentos complexos e skills.This método pode ser aplicável a outras espécies de mamíferos.

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

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

Um novo método para obter macrófagos de cultura primária de células de fígado de rato é descrito. Este método utiliza a proliferação de macrófagos na cultura, seguido por agitação de frascos de cultura e purificação pelo apego seletivo para pratos de plástico. Esta técnica proporciona eficiente macrófagos do fígado, sem equipamentos complexos e habilidades.

Um método simples e eficiente para isolar Macrófagos de Mixed culturas primárias de células do fígado Adulto

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

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Immunology and Infection

37.9K visualizações.  National Institute of Agrobiological Sciences, Tsukuba, Japan. Um novo método para obter macrófagos de cultura primária de células de fígado de rato é descrito. Este método utiliza a proliferação de macrófagos na cultura, seguido por agitação de frascos de cultura e purificação pelo apego seletivo para pratos de plástico. Esta técnica proporciona eficiente macrófagos do fígado, sem equipamentos complexos e habilidades.

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

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

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

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

Geração de Culturas Raft organotípicas de queratinócitos humanos primários

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

Imunologia e Infecção

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 ...

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

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

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

Um método simples e eficiente para detectar ativação do fator nuclear em neutrófilos humanos por citometria de fluxo

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

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

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

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

A transfecção altamente eficiente de THP-1 por macrófagos humanos Nucleofection

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

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

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

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

Um método de rendimento eficiente e alta para o isolamento de subpopulações de células dendríticas Rato

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

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

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

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

Uma abordagem simples e eficiente para construir vectores de vírus mutante Vaccinia

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

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

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

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

Geração e identificação de GM-CSF macrófagos derivados e dendríticas células alveolares do tipo de ratinho de Medula Óssea

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

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

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

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

A indução de uma resposta inflamatória em culturas primárias de hepatócitos de ratinhos

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

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

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

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

Uma maneira não invasiva de isolar e células de fenótipo da Conjuntiva

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

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

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

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

Um método simples e eficiente para estabelecer linhas de células T de pacientes com infecção crônica ativa vírus Epstein - Barr e NK

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