Gostaríamos de agradecer aos membros do laboratório Tsirka para os seus conselhos e comentários úteis. Este trabalho foi apoiado por R01NS42168 a SET, 12PRE12060489 a RB, um NSF-3MT IGERT e uma dissertação comunhão Turner a LT, e NSF-3MT IGERT de JCN.

1. Dissecção (Dia 0) <ol><li> Induzir a anestesia para P0-2 filhotes de rato com hipotermia. Limpe as cabeças dos filhotes com um Kimwipe embebido em álcool 70% para desinfecção do tecido. </li><li> Retire as cabeças com um par de tesouras, usando quatro filhotes por 10 centímetros placa de cultura. Coloque as cabeças em uma placa de Petri contendo tampão de gelada Hank. </li><li> Ancorar as cabeças usando uma pinça curva através das órbitas dos olhos e remova cuidadosamente a pele que cobre o crânio com micropinças retas. </li><li> Retire os ossos do crânio com micropinças em linha reta, tendo o cuidado para não perfurar ou danificar o córtex. A maneira mais eficaz é a iniciar a remoção de partida perto do cerebelo, a qual está localizada na base da cabeça, que esta região será descartado. </li><li> Remover o cérebro, com uma pequena espátula, e colocar os (4) os cérebros numa placa de petri fresca contendo solução tampão gelada de Hank. </li><li> Todas as etapas deste ponto em diante utilizar micropinças e são realizadas sob a divulmicroscópio cção. Começando no lado ventral do cérebro, ancorar o tecido, mantendo o cerebelo e fazer duas pequenas incisões em cada lado do mesencéfalo. Tenha cuidado para não cortar todo o caminho através do tecido, como o córtex cobrir o mesencéfalo nesta região. </li><li> Gentilmente provocar o mesencéfalo e cerebelo em um pedaço das duas corticais. Os dois córtices deve formar uma forma côncava. </li><li> Separe as duas corticais e orientar um único córtex com o lado medial para posterior dissecação. O hipocampo pode ser difícil observar, mas está localizado no lado oposto do bulbo olfactivo, que aparece como um nódulo pequeno na extremidade pontiaguda do córtex. </li><li> Remover o hipocampo, que tem uma forma de crescente. Virar o córtex mais para ver do lado dorsal. </li><li> Usando o bolbo olfactivo como um ponto de partida, remover todas as meninges e o próprio bulbo olfactivo do córtex. </li><li> Os córtices dissecados devem ser colocados em tubos cónicos de 15 ml com 14 ml de colsolução salina tamponada de Hank d em gelo. </li></ol> 2. Cultura de Células (Dia 0) <ol><li> Pré-revestimento de placas de 10 cm de cultura de tecido com 5 ug / ml de poli-D-lisina (PDL) diluído em água esterilizada durante 3 horas a 37 ° C. </li><li> Aspirar PDL, e as placas de lavagem uma vez com água autoclavada. Secar sob a placa de UV no capuz de cultura de tecidos durante 20 min. Este passo deve ser realizado imediatamente antes do processo de esvaziamento. </li><li> Aspirar o tampão de Hank de tubos de ensaio contendo os córtices a partir de 4 cérebros cada, aplicar 4 ml de solução de tripsina / EDTA 1x, tritura-se o tecido com p1000 ponta, e coloque os tubos em 37 ° C incubadora durante 15 min. </li><li> Adicionar 4 ml de mídia microglia completos por tubo para parar a digestão enzimática, misturar e spin para baixo conteúdo em 1.5K rpm por 5 min. </li><li> Aspirar o sobrenadante e repetir lavagem com 4 ml de meio completo. Suspenda as células em 10 ml de meio completo microgliais (DMEM com 10% de FBS, piruvato de sódio a 1%, 0,08% de gentamicina), e através de um filtro de 40 micron filtro de células da malha. </li><li> Placa a uma densidade de 8 córtices por 10 ml de uma placa de cultura de 10 cm de tecido e colocar num incubador de cultura de tecido a 37 ° C e 5% de CO 2 (Ver Adulto Microglia protocolo). </li></ol> (Dia 3) <ol><li> Mudança de mídia em todos os pratos de cultura de células com a mídia microglia completos. </li></ol> (Dia 10) <ol><li> Adicionar 400 microlitros de 60 mM de lidocaína em HBSS (para separar microglia) em meio de placas de cultura de tecidos de 10 cm e incubar à temperatura ambiente (RT) durante 10-15 min. </li><li> Recolher os meios de suspensão / célula a partir da placa, e lava-se a placa uma vez com tampão de Hank. Recolher o tampão de lavagem para recuperar restante da microglia. </li><li> Adicionar EDTA 5 mM (pH 8,0) à suspensão de células até uma concentração final de 50 mM ou a uma diluição de 1/100. </li><li> Girar suspensão de células a 1000 x g (1500 rpm) durante 5 minutos e re-suspensão em 1 ml de DMEM com FBS a 1%. </li><li> Contar o número de células viáveis ​​(como por Mi Adultocroglia 3.1/3.2) e células divididas em placas de cultura de tecidos na densidade experimental desejado. Aproximadamente 1x10 6 microglia são colhidas a partir de uma placa de 10 cm contendo os córtices a partir de 4 cérebros. Para imunofluorescência, recomenda-se uma densidade de 2.5x10 4 células por 18 milímetros lamela. </li><li> Deixe pelo menos 24 horas para micróglias para voltar totalmente ao seu ramificada, estado de repouso antes de usar. </li></ol> Protocolo Texto: (Adult Microglia) 1. Coleção de tecidos <ol><li> Placas de cultura de tecidos de revestimento com poli-D-lisina (PDL), durante 3 horas a 37 ° C ou durante a noite a 4 ° C. Lavar as placas uma vez com água esterilizada, imediatamente antes de usar. </li><li> Eutanásia rato por asfixia CO 2 e verificar que a eutanásia é completa. Limpe a área da medula espinhal com etanol 70%. </li><li> Utilizando um par de tesouras de cortar a pele na parte superior da medula espinhal; então proceder para cortar a coluna vertebral a partir da região T1 a T12. Cortaro músculo restante fora dos lados da medula espinal. </li><li> Lentamente cortar as vértebras usando microtesoura como você segurar delicadamente a medula espinhal com os dedos. Seja o cuidado de não perfurar a medula espinhal, como o tecido é muito suave. Lentamente extrair a medula espinal utilizando microfórceps. </li><li> Submerge da medula espinhal em uma placa de Petri contendo HBSS gelada. Usando um microscópio de luz remover todas as meninges visíveis usando micropinças </li><li> Corte da medula espinhal em segmentos transversais pequenas o suficiente para gerar vários fragmentos. A espessura dos fragmentos não deve ser superior a 2 mm a fim de facilitar uma digestão eficiente. Transferir as peças da medula espinal para um tubo de 15 ml contendo 1 ml cónico de HBSS gelada. Manter o tubo em gelo até à etapa de digestão. </li></ol> 2. A digestão de tecidos <ol><li> Aspirar HBSS de 15 ml tubo cônico contendo tecido da medula espinhal. Tenha cuidado para não aspirar qualquer pedaço de tecido. </li><li> Adicionar 1 ml de tripsina (2.5 g / L de tripsina irradiada porcino e 0,2 g / L de EDTA em HBSS) e incubar a 37 ° C durante 30 min. </li><li> Para parar a digestão remover a tripsina e adicionar 3 ml de meio de microglia primário que contém soro de travar a digestão enzimática. </li><li> Pipeta cima e para baixo para retirar o tecido. Filtra-se a suspensão de células através de um filtro de 40 um da célula. </li></ol> 3. Contagem de células e chapeamento <ol><li> Misturar um volume igual de suspensão de células e solução DAPI. </li><li> Contar as células utilizando um hemocitómetro. </li><li> Placa com uma densidade de entre 5-7x10 5 células / ml em PDL revestidos 35 x 10 mm pratos. Galvanização a uma densidade mais baixa impediu o crescimento celular, mesmo quando as células foram cultivadas em placas de 12 poços que têm uma área menor em comparação com 35 x pratos de 10 mm. </li></ol>

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Os autores não têm nada a revelar.

A microglia modular CNS funcionamento normal, bem como as respostas inflamatórias a diversas patologias. Funcional remodelação sináptica por microglia tem sido implicado na manutenção da homeostase do cérebro normal 15. Durante a cascata neurogênica participam na depuração de células progenitoras neurais do giro denteado do hipocampo 4, 16. Por conseguinte, é necessário desenvolver um sistema de cultura no qual a estudar microglia neonatal e adulto, que vai cobrir a vasta área do dese...

A microglia são as células imunitárias residentes do SNC, que mais se assemelham macrófagos periféricos em estrutura e função 1. Foi recentemente demonstrado que as células microgliais pós-natais derivar de progenitores mielóides primitivos e são gerados antes do oitavo dia da embriogênese erigir a noção anterior que progenitores hematopoiéticos pós-natais são a fonte da microglia no cérebro adulto 2. Eles desempenham um papel-chave em várias doenças neurológicas e pode responder rapidamente a uma infecção ou lesão, liberando citocinas pró-inflamatórias ou anti-inflamatória 3. Assim microglia abranger uma unidade autónoma no SNC, que podem ser manipuladas para afectar a progressão da doença. Desenvolvimento de métodos robustos e reprodutíveis para isolar e cultivar células microgliais recém-nascidos ou adultos é importante para estudos futuros. Microglia é conhecido por ser jogadores críticos de um número de patologias cerebrais. Mais recentemente, o roles são emergente para as células em desenvolvimento e a função normal do cérebro, como microglia fagocitam excesso de células progenitoras neurais a partir de giro dentado do hipocampo de 1, 4. Microglia também pode modular várias condições neurológicas que afetam a medula espinhal, tais como MS, dor neuropática, e lesão medular 5-7. Microglia da medula espinal reagem de forma diferente em relação ao cérebro microglia em resposta a sinais de ativação 8, 9, provavelmente devido às diferenças no ambiente local. Assim, é importante estabelecer um adequado sistema in vitro para estudar a cultura e microglia espinal medula. Microglia Neonatal produzir significativamente mais do óxido nítrico citocina pró-inflamatória em comparação com células adultas após estimulação in vitro com o IFN-γ ou TNF-a, 10,11 destacando ainda mais a necessidade de utilizar células adultas para o estudo da microglia no contexto de certas doenças. O protocolo que empregamos no laboratório de cuLTURE microglia neonatal é uma variação dos métodos recentes que utilizam a agitação de culturas mistas de glia, num esforço para remover a microglia da superfície do frasco de cultura de célula 12. Também descrevemos um método de cultura da microglia de ratinho adulto a medula espinal por meio do protocolo descrito pela primeira vez por Yip et ai 13. Este método proporciona uma maneira mais rápida de células adultas de cultura em comparação com outros protocolos disponíveis 14. A preparação resultante é de 70% a microglia, sendo a percentagem restante é constituída por astrócitos. Embora o grau de pureza da nossa cultura é menor em comparação com outros métodos publicados 13, este sistema de cultura é útil para explorar a microglia cultura em resposta a vários estímulos de activação, bem como para o estudo de doenças que afectam principalmente a medula espinal e no qual um forte resposta inflamatória é uma característica principal. Todos os protocolos descritos foram aprovados pelo Stony Brook University IACUC.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		 </tr>
		 <tr>
			<td>EDTA, 5mM</td>
			<td>Invitrogen</td>
			<td>15567-028</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lidocaine, 60mM</td>
			<td>Sigma</td>
			<td>L-5647</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypan Blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypsin/EDTA</td>
			<td>Cellgro</td>
			<td>25-052-CI</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM, 1X</td>
			<td>Cellgro</td>
			<td>10-017</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium Pyruvate</td>
			<td>Cellgro</td>
			<td>25-000-Cl</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gentamycin Sulfate</td>
			<td>Biowittaker</td>
			<td>17-518Z</td>
			<td></td>
		 </tr>
		 <tr>
			<td>35 x 10mm tissue culture dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Poly-D-Lysine, 100 &mu;g/ml</td>
			<td></td>
			<td></td>
			<td>Dilute 1:20</td>
		 </tr>
		 <tr>
			<td>HBSS, 1X</td>
			<td>Cellgro</td>
			<td>20-023-CV</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

culturing microglia from the neonatal and adult central nervous system

A microglia residentes são as células de macrófagos, como do sistema nervoso central (SNC) e, como tal, têm um papel extremamente importante nos processos fisiológicos e patológicos, tais como a maturação do SNC em desenvolvimento, a esclerose múltipla, e lesão da medula espinal. Microglia podem ser ativados e recrutado para a ação por lesão neuronal ou estimulação, como dano axonal visto em MS ou trauma cerebral isquêmica resultante de acidente vascular cerebral. Estes membros imunocompetentes do SNC também são pensados ​​para ter papel na plasticidade sináptica em condições não patológicas. Empregamos protocolos para microglia cultura de tecidos neonatais e adultos que são destinadas a maximizar o número de células viáveis, minimizando variáveis ​​interferentes, tais como a presença de outros tipos de células do sistema nervoso central e os restos de cultura de células. Nós utilizamos grandes e facilmente discernível componentes do SNC (por exemplo, do córtex, os segmentos da medula espinal), o que torna todo o processo viável e reprodutível. O uso da célula adultas uma alternativa adequada para o uso de neonatal microglia do cérebro, como muitas patologias estudados afectam principalmente a medula espinal pós-natal. Esses sistemas de cultura são também úteis para testar directamente o efeito dos compostos que podem inibir ou estimular a activação da microglia. Uma vez que a activação da microglia, podem moldar os resultados da doença no SNC adulto, existe uma necessidade de sistemas in vitro em que a microglia neonatal e adulto podem ser cultivadas e estudadas.

<html> Um exemplo de células microgliais de repouso e activado é mostrado na Figura 1. A microglia foram visualizadas 24 horas após o plaqueamento (Figura 1a) e exibem morfologia ramificada (de repouso). A exposição ao reagente de iniciação, o lipopolissacarídeo bacteriano (LPS), resulta em alterações na morfologia da microglia, como as células ficam activadas (Figura 1b). Um exemplo de contagem das células de revestimento é most...

Watch this Scientific Journal Video about Culturing Microglia from the Neonatal and Adult Central Nervous System at JoVE.com

Culturing Microglia from the Neonatal and Adult Central Nervous System

Nós delinear métodos para a rápida e eficiente isolamento / cultura da microglia viável a partir do córtex cerebral neonatal e adulto medula espinhal. A dissecção e revestimento de microglia cortical pode ser realizado dentro de 90 minutos, com a colheita da microglia subseqüente ocorrendo ~ 10 dias após a dissecção inicial.

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 ...

culturing-microglia-from-the-neonatal-and-adult-central-nervous-system

Research

JoVE Journal

Immunology and Infection

27.6K visualizações.  Stony Brook University. Nós delinear métodos para a rápida e eficiente isolamento / cultura da microglia viável a partir do córtex cerebral neonatal e adulto medula espinhal. A dissecção e revestimento de microglia cortical pode ser realizado dentro de 90 minutos, com a colheita da microglia subseqüente ocorrendo ~ 10 dias após a dissecção inicial.

Vídeo: Culturing Microglia from the Neonatal and Adult Central Nervous System

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

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Purificação e visualização de lipopolissacarídeo de bactérias Gram-negativas por extracção aquosa de fenol-Hot

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

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 ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Desenvolvimento de um Sistema Reporter vírus da hepatite B de acompanhar as fases iniciais do ciclo de replicação

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

Análise de Microglia e Macropagos derivados de Monócitos do Sistema Nervoso Central por Citometria de Fluxo

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Isolando os linfócitos de rato pequeno Intestinal sistema imunológico

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the central nervous system (CNS). Recent studies demonstrate the use of a peptide, RVG9R, incorporating the minimal receptor-binding domain of the Rabies virus glycoprotein, in eliciting the delivery of siRNA into neurons in the brain. In this protocol, the peptide-siRNA formulation is delivered intranasally with a pipette in the dominant hand, while the anesthetized mouse is restrained by the scruff with the nondominant hand in a "head down-and-forward position" to avoid drainage into the lung and stomach upon inhalation. This precise gripping of mice can be learned but is not easy and requires practice and skill to result in effective CNS uptake. Furthermore, the process is long-drawn, requiring about 45 min for the administration of a total volume of ~20-30 &#181;L of solution in 1-2 &#181;L droplet volumes per inhalation, with 3-4 min rest periods between each inhalation. The objective of this study is to disclose a mouse positioning device that enables the appropriate placement of mice for efficient IN administration of the peptide-siRNA formulation. Multiple features are incorporated into the design of the device, such as four or eight positioning chairs with adjustable height and tilt to restrain anesthetized mice in the head down-and-forward position, enabling easy visualization of the mice's nares and a built-in heating pad to maintain the mice's body temperatures during the procedure. Importantly, the ability to treat four or eight mice simultaneously with RVG9R-siRNA complexes in this manner enables studies on a much quicker time scale, for the testing of an IN therapeutic siRNA approach. In conclusion, this device allows for appropriate and controlled mouse head positioning for IN application of RVG9R-siRNA and other therapeutic molecules, such as nanoparticles or antibodies, for CNS delivery.

Here, we present a protocol using a mouse positioning device that enables the appropriate placement of mice for the intranasal administration of a brain-targeting peptide-siRNA formulation enabling effective gene silencing in the central nervous system.

Um dispositivo de posicionamento para a colocação de camundongos durante a entrega de siRNA intranasal ao sistema nervoso central

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Análise Citométrica de Fluxo de Infiltração linfócito no Sistema Nervoso Central durante Encefalomielite Autoimune Experimental

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

Isolando Tecidos do Sistema Nervoso Central e Meninges Associadas para Análise a Jusante de Células Imunes

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...

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

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

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

Protocolo Econômico e Eficiente para Isolar e Culturing Células Dendríticas Derivadas da Medula Óssea de camundongos

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