Financiado pelo programa intramural na Universidade Militar.

1. Dissecção da Tissue Neonatal cérebro de rato <ol><li> L-15 de Leibovitz frio meios condicionados (Leibowitz L-15 + 0,1% de BSA + Pen 1% / Strep) a 4 ° C. Meios de cultura quentes (DMEM + 10% FBS + 1% de penicilina / estreptomicina) para 37 ° C. Prepare 4 60x15 mm pratos de Petri com 4-5 ml de L-condicionado de 15 meios de comunicação sobre o gelo. Esterilizar todos os instrumentos cirúrgicos com 100% de etanol. </li><li> Lavar as P2 filhotes rap neonatais com etanol 70%. (Crias de ratos neonatais com idades compreendidas entre P1-P5 pode ser usado neste protocolo.) </li><li> Rapidamente decapitar um rato filhote com uma tesoura afiada estéreis e soltar a cabeça imediatamente em etanol 70%. Repetir para um total de 5 crias de rato, em seguida, transferir cabeças em solução salina. </li><li> Remover os cérebros inteiros das cabeças e coloque em uma placa de Petri com 4 - 5 ml de solução de L-15 (Leibowitz L-15 + 0,1% de BSA + 1% Pen / Strep) em gelo. </li><li> Repita os procedimentos 1.2 - 1.4 para os filhotes restantes no lixo. </li><li> Remover as meninges a partir do cérebro e transferir o destecido cerebral IRED (isto é, córtex, cerebelo, cérebro inteiro, etc) em uma placa de Petri de novo com 4-5 ml de solução de L-15 (Leibowitz L-15 + 0,1% de BSA + Pen 1% / Strep) em gelo. </li></ol> 2. Preparação de população mista de células da glia <ol><li> Sem o tecido cerebral prejudicial com uma tesoura ou uma lâmina, transferir o tecido com uma pipeta de 10 ml para um tubo estéril de 50 ml cónico. </li><li> Centrifugar a 2.500 cónica RCF durante 5 min a 4 ° C. </li><li> Aspirar o sobrenadante. Adicionar ml de 4-5 frescos meio L-15 (Leibowitz L-15 + 0,1% de BSA + Pen 1% / Strep). </li><li> Pipetar tecido cima e para baixo 10 vezes com uma pipeta de 10 ml estéril. </li><li> Coloque um filtro celular (100 poros mm) para um novo tubo de 50 ml. Pipetar tecido cima e para baixo uma vez com uma solução estéril 5 ml pipeta e com a pipeta de descarga para o filtro de células, dispensar o material através do filtro de célula para dentro do tubo cónico. </li><li> Lavar o filtro de células com 4-5 ml de frescos meio L-15 (Leibowitz L-15 + BS 0,1%Um Pen + 1% / Strep). </li><li> Centrifugar cónica com células tensas a 2.500 RCF durante 5 min a 4 ° C. </li></ol> 3. Chapeamento e manutenção da cultura mista de células gliais <ol><li> Para cada cérebro de rato pup processado, preparar 1 T-75 estéreis do balão por adição de 12 ml de meio de cultura (DMEM + 10% FBS + 1% de penicilina / estreptomicina) em cada frasco. </li><li> Aspirar o sobrenadante das células peletizadas e adicionar 5-6 ml de meio de cultura para o sedimento de células. Pipetar cima e para baixo 10 vezes com uma pipeta de 10 ml. </li><li> Transferir um volume igual de suspensão de células a cada balão T-75. </li><li> Incubar em frascos de 5% de CO 2 incubadora a 37 ° C durante um total de 1-3 semanas. </li><li> Frascos deve ser primeiro incubadas durante 5 dias, e no quinto dia, substituir os meios de cultura em cada balão com 12 ml de meio fresco e retornar para a incubadora. Em seguida, a cada 3 dias, substituir os meios condicionados em cada balão com 12 ml de meio fresco para atingir confluência. Isso deve ser fazerne com muito cuidado, sem tocar no fundo dos frascos, onde as células se ligam. </li></ol> 4. Isolamento e Chapeamento de Microglia primária <ol><li> Após cultura mista de gliais são completamente confluentes, remover frascos da incubadora e cobrir as tampas recipiente com parafilme para evitar a troca gasosa com o ar ambiente. </li><li> Agitar frascos de 100 RPS (Lab Companion SI-600, Jeio Tech) durante 1 hora a 37 ° C. </li><li> Collect meios de comunicação de todos os frascos com uma pipeta de 10 ml sem perturbar a camada de astrócitos na superfície balão. Coloque a mídia em tubos de 50 ml. Adicionar meios frescos aos frascos e frascos de retorno para a incubadora. </li><li> Centrifugar a 2.500 tubos cónicos RCF durante 5 min a 4 ° C. </li><li> Aspirar o sobrenadante a partir de todos os tubos cónicos. Peletes de células são células da microglia de alta pureza. Ressuspender pelotas em 1 ml de plaqueamento microglia media (DMEM + FES 10% + 1% de penicilina / estreptomicina). </li><li> Contar a densidade celular nos meios de comunicação que utilizam ressuspensosum hemocitómetro. </li><li> Adicionar um volume apropriado de meios de revestimento microglia para atingir um 2 x 10e5 células por ml de densidade. Placa conforme for apropriado para análise experimental. </li><li> Permitir microglia para anexar durante a noite. </li></ol> 5. Os resultados representativos O protocolo descrito acima resulta em alta pureza culturas primárias microglia. Como determinado por imuno-histoquímica para um marcador de células de macrófagos / microglia específico (Iba1), as culturas são chapeadas microglia&gt; puro a 90% (Figura 2). Além disso, a coloração para os marcadores de células de astrócitos, oligodendrócitos e neurónio específicos demonstra a contaminação mínima (Figura 2). Depois de estabelecer cultura mista de células gliais, microglia pode ser isolado por agitação até 4 vezes sucessivas. O isolamento inicial microglia irá produzir aproximadamente 2,2 x 106 células / ml ou cerca de 9 milhões de células por 10 frascos eo rendimento esperado diminui com cada sha sucessivake. Microglia isolado por este método têm sido utilizados com sucesso para investigar a capacidade de fagocitose, função, tais como a produção de óxido nítrico, e toxicidade neuronal (Figura 3) 13-14. <img alt="A Figura 1" src="/files/ftp_upload/3814/3814fig1.jpg" /> Figura 1. Visão geral do protocolo para a preparação de mistura de culturas de células gliais e de isolamento da microglia primária. <img alt="A Figura 2" src="/files/ftp_upload/3814/3814fig2.jpg" /> Figura 2. Isolamento de microglia alta pureza como verificado pela análise imuno-histoquímica usando microglia (Iba-1, vermelho), astrócitos (GFAP, verde), neurônio (NeuN, vermelho) e oligodendrócitos (CC1, vermelho) marcadores específicos. A coloração para DNA de células em cultura por DAPI (azul) é também mostrada. <img alt="A Figura 3" src="/files/ftp_upload/3814/3814fig3.jpg" /> Figura 3. Microglia isolado no método descricama têm sido usados ​​em vários ensaios, incluindo, mas não se limitam a, ensaios de fagocitose, ensaios funcionais e estudos de neurotoxicidade. Nestes estudos, verificou-se que a microglia (Iba-1 positivo, verde), quando expostos a controlar (A) ou LPS (B) meios de comunicação tratados, aumentar a sua fagocitose de pérolas de látex marcadas por fluorescência (vermelho), que pode ser quantificado ( C). Além disso, LPS de tratamento (1ng/ml) pode aumentar a produção de óxido nítrico em microglia (D). Finalmente, através de ambos transwell inserto-separadas ou directos microglia-neuronais culturas, nós descobrimos que a microglia incubadas com LPS pode induzir a morte de células neuronais, como medido por lactato desidrogenase (LDH) de libertação (E, F).

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Embora este protocolo é rotineiramente utilizado para produzir microglia puro e saudável para experimentos de pesquisa, uma análise cuidadosa dos aspectos técnicos, durante partes críticas do procedimento minimizar a variabilidade na microglia isolado. Primeiro, durante a dissecção do tecido do cérebro de filhotes de ratos neonatos, trabalhando em tempo hábil é necessário para minimizar os danos e hipóxico isquêmica do tecido. No entanto, é também importante para remover completamente o revestimento mení...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentários </td></tr><tr><td> 60 mm x 15 mm, placas de Petri </td><td> Fisher marca </td><td> 0875713A </td><td></td></tr><tr><td> Afiadas tesouras de dissecação </td><td> Belas Ferramentas Ciência </td><td> 14094-11 </td><td></td></tr><tr><td> Dumont # 7b fórceps padrão pontas, curvas, 11cm </td><td> Belas Ferramentas Ciência </td><td> 11270-20 </td><td></td></tr><tr><td> Dumont # 5 fórceps padrão dicas, em linha reta, 11 cm </td><td> Belas Ferramentas Ciência </td><td> 11251-10 </td><td></td></tr><tr><td> Cónico de 50 ml de tubos de centrifugação </td><td> VWR </td><td> 89039-656 </td><td></td></tr><tr><td> 5 ml pipetas sorológicas </td><td> Grenier Bio One </td><td> 606180 </td><td></td></tr><tr><td> 10 ml pipetas sorológicas </td><td> Grenier Bio One </td><td> 607180 </td><td></td></tr><tr><td> 100 filtro célula mM estéril nylon </td><td> Falcão </td><td> 35-2360 </td><td></td></tr><tr><td> 75 cm 2 de cultura de tecidos frascos </td><td> Corning </td><td> 430641 </td><td></td></tr><tr><td> Meio mínimo essencial de Dulbecco (DMEM) </td><td> Gibco (Invitrogen) </td><td> 31053-028 </td><td></td></tr><tr><td> L-15 de Leibovitz médio </td><td> Gibco (Invitrogen) </td><td> 11415064 </td><td></td></tr><tr><td> Soro fetal de bovino </td><td> Gibco (Invitrogen) </td><td> 16000-036 </td><td></td></tr><tr><td> Fetal soro eqüino </td><td> Pescador </td><td> SH3007402 </td><td></td></tr><tr><td> Penicilina-Estreptomicina </td><td> Gibco (Invitrogen) </td><td> 15140163 </td><td></td></tr><tr><td> Etanol a 100% </td><td> A Warner Empresa Graham </td><td> 64-17-5 </td><td></td></tr><tr><td> Solução salina tamponada com fosfato, 10X, pH 7,4 </td><td> Qualidade Biológica, inc. </td><td> 119-069-131 </td><td> 1X em água destilada estéril </td></tr><tr><td> Sacos de Biohazard </td><td> VWR </td><td> 14220-028 </td><td></td></tr><tr><td> Hemocitómetro </td><td> Hausser Científico </td><td> 1492 </td><td></td></tr><tr><td> 6-poços de células placas de cultura com a superfície Cellbind </td><td> Corning </td><td> 3335 </td><td></td></tr></tbody></table>

primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue

Microglia conta para aproximadamente 12% do total da população celular no cérebro de mamíferos. Embora os neurónios e astrócitos são considerados os tipos de células principais do sistema nervoso, microglia desempenhar um papel significativo na fisiologia normal do cérebro através da monitorização do tecido para que os detritos e os agentes patogénicos e manutenção da homeostase do parênquima através 1,2 actividade fagocítica. A microglia é ativada durante uma série de lesões e doença, incluindo doenças neurodegenerativas, lesões cerebrais traumáticas e infecção do sistema nervoso 3. Sob estas condições de ativação, microglia aumentar sua atividade fagocitária, passam por mudanças morpohological e proliferativa, e ativamente secretam espécies reativas de oxigênio e nitrogênio, pró-inflamatórias quimiocinas e citocinas, muitas vezes, ativando um loop parácrina ou autócrina 4-6. Como essas respostas microgliais contribuir para a patogênese da doença em condições neurológicas, a pesquisa focada em microglia se justifica. Devido à heterogeneidade celular do cérebro, é tecnicamente difícil a obtenção de material de amostra suficiente microglial com pureza elevada durante experiências in vivo. A pesquisa atual sobre as funções neuroprotetoras e neurotóxico de microglia exigir um método de rotina técnica para gerar consistentemente microglia puro e saudável, com rendimento suficiente para o estudo. Apresentamos, no texto e vídeo, um protocolo para isolar microglia primário puro a partir de culturas da glia mistos para uma variedade de aplicações a jusante. Resumidamente, esta técnica utiliza tecido cerebral dissociada crias de rato neonatal para produzir mistos culturas de células gliais. Após as culturas mistas gliais atingir a confluência, microglia primárias são mecanicamente isolado a partir da cultura por um breve duração de agitação. Os microglia são então plaqueadas a elevada pureza para o estudo experimental. O princípio e protocolo desta metodologia ter sidodescrito na literatura 7,8. Além disso, as metodologias alternativos para isolar microglia primários estão bem descritos 9-12. Tecido cerebral homogeneizado podem ser separados por centrifugação em gradiente de densidade para produzir microglia primário 12. No entanto, a centrifugação é de comprimento moderado (45 min) e pode causar danos celulares e activação, assim como, causar enriquecido microglia e outras populações celulares. Outro protocolo tem sido utilizada para isolar a microglia primária em uma variedade de organismos por prolongada (16 horas), agitando ao mesmo tempo em cultura 9-11. Após agitação, o sobrenadante é centrifugado meios para isolar a microglia. Este método de isolamento de mais duas etapas também podem perturbar a função e ativação da microglia. Nós principalmente utilizar o protocolo de isolamento seguinte microglia no nosso laboratório para um número de razões: (1) primária microglia simular em biologia in vivo mais fielmente que imortalizadas linhas celulares de roedores microglia, (2) Uma ruptura nominal mecânica minimiza disfunção celular potencial ou activação, e (3) o rendimento suficiente pode ser obtida sem passagem das culturas mistas de células gliais. É importante notar que este protocolo utiliza o tecido cerebral de crias de rato neonatal para isolar microglia e que a utilização de ratos mais velhos para isolar microglia pode ter um impacto significativo no rendimento, estado de activação, e as propriedades funcionais da microglia isolado. Há evidências de que o envelhecimento está relacionada com a disfunção microglia, neuroinflamação aumentada e patologias neurodegenerativas, assim estudos anteriores têm utilizado ex vivo adulto microglia para compreender melhor o papel da microglia em doenças neurodegenerativas, onde o envelhecimento é parâmetro importante. No entanto, ex vivo microglia não pode ser mantido em cultura durante períodos prolongados de tempo. Consequentemente, embora este protocolo prolonga a vida da microglia primária em cultura, deve notar-se que a microglia comportar differently de adulto microglia e estudos in vitro deve ser cuidadosamente considerado quando traduzido para uma definição em vivo.

Watch this Scientific Journal Video about Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue at JoVE.com

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

Isolar microglia primária a partir da heterogeneidade celular do cérebro é essencial para investigar o seu papel em ambas as condições fisiológicas e patológicas. Este protocolo descreve um isolamento mecânico e técnica de cultura mista de células que fornece alto rendimento e elevada pureza, viáveis ​​primárias células microgliais para<em&gt; In vitro</em&gt; Estudo e aplicações a jusante.

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 ...

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JoVE Journal

Immunology and Infection

39.1K visualizações.  Uniformed Services University. Isolar microglia primária a partir da heterogeneidade celular do cérebro é essencial para investigar o seu papel em ambas as condições fisiológicas e patológicas. Este protocolo descreve um isolamento mecânico e técnica de cultura mista de células que fornece alto rendimento e elevada pureza, viáveis ​​primárias células microgliais para In vitro Estudo e aplicações a jusante.

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

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

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

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 ...

Imunologia e Infecção

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 ...

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 of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (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 and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Isolamento e injecção intravenosa de Murino de Medula Óssea Derivado monócitos

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

Isolamento de células de Sertoli e as células de rato peritubular Testículos

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Isolamento e análise de citometria de fluxo de células imunes do cérebro isquêmico Rato

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

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 ...

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-&#954;B (NF-kB) and inhibitor &#954;B (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

Here, we present a protocol to isolate brain nuclei in the neonatal rat brain in conjunction with first colostrum feeding. This technique allows the study of nutrient insufficiency stress in the brain as modulated by enterocyte signaling.

Avaliação de estresse celular e inflamação em discreta secretoras de ocitocina núcleos de cérebro em ratos Neonatal antes e depois da primeira mamada do colostro

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis.

The overall goal of this protocol is to instruct how to extract, maintain, and dissociate murine astrocyte and microglia cells from the central nervous system, followed by infection with protozoa parasites.

Isolamento concomitante de astrócitos primários e microglia para infecção por parasitas protozoários

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central ...