Name: the use of mouse splenocytes to assess pathogen-associated molecular pattern influence on clock gene expression
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Description: Watch this Scientific Journal Video about The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression at JoVE.com

Este trabalho foi apoiado pela pesquisa de faculdade concede do escritório da faculdade de artes e Ciências do reitor na Universidade de Hartford.

Durante o estudo, tratamento e cuidados com animais cumprido com a política do National Institutes of Health, estavam em conformidade com as orientações institucionais e foram aprovados pelo Comitê de uso e cuidado Animal institucional Animal Universidade de Hartford.
1. o arrastamento de animais
Nota: Vinte semanas masculino B6129SF2/J os ratos são usados no estudo.
<ol><li>Arrastar os ratos para um 12 h luz (padrão sobrecarga luz branca) / ciclo de 12h escuro durante 2 semanas antes do experimento. Nota: Aqui tempo zeitgeber (ZT) 0 corresponde a luzes acesas e ZT12 de luzes apagadas, mantendo todos os outros fatores ambientais (ou seja, comida, água e temperatura) constante 21.</li>
</ol>2. preparação dos instrumentos, meio de cultura e meio de desafio
<ol><li>Autoclave fórceps e a tesoura de dissecação. Enrole pares de corrediças do microscópio fosco em folha de alumínio e autoclave.</li>
	<li>Prepare o meio de cultura por adição de soro fetal bovino (FBS) para RPMI 1640 para uma concentração final de 10%. Prepare-se 10 mL de meio de desafio em tubos de 50 mL, adicionando os seguintes PAMPs ao meio de cultura (RPMI com 10% FBS); LPS (5 µ g/mL), ODN1826 (5 µ g/mL), calor-matou Listeria monocytogenes (108 HKLM/mL), ou outro PAMP em sua concentração sugerida.</li>
	<li>Aquecer o meio de cultura e meio de desafio a 37 ° C em banho-maria e manter cerca de 70 mL de soro fisiológico estéril tamponado de fosfato (PBS, pH 7,2) à temperatura ambiente.</li>
	<li>Adicione 10 mL de meio de cultura usando uma pipeta de 10 mL e pipeta ajuda para um tubo de 50 mL e lugar no gelo.</li>
	<li>Adicione aproximadamente 30 mL de etanol a 70% para fins de copo e coloque um 100ml de tesoura e pinça de dissecação no copo medidor para evitar a contaminação microbiana.</li>
	<li>Prepare-se do lysis para isolamento de RNA adicionando 10 µ l de β-mercaptoetanol (sob uma coifa) para cada 1 mL de tampão RLT em um tubo de 50 mL. Faça apenas a quantidade que será necessária (600 µ l por amostra, que consiste em aproximadamente 1 x 106 células). Nota: O Buffer RLT é um componente do kit de extração de RNA que suporta a ligação do RNA para a membrana de silicone.</li>
</ol>3. Splenocyte isolamento e desafio
<ol><li>Eutanásia em ratos a um momento de particular zeitgeber através de narcose por mantê-los em sua gaiola original e adição de CO2 para a gaiola, a uma taxa de fluxo de 3 L/min. Continue fornecendo CO2 por 1 min após a respiração para.</li>
	<li>Confirme a morte através do deslocamento cervical, colocando o polegar e o dedo indicador em ambos os lados do pescoço na base do crânio. Alternativamente, pressione uma haste na base do crânio enquanto puxa rapidamente (usando a outra mão) a base da cauda ou membros posteriores para causar a separação das vértebras cervicais do crânio22.</li>
	<li>Pulverize o tronco de rato com etanol a 70% e limpe com uma toalha de papel. Posicione o mouse na sua traseira e ligeiramente inclinada para o seu lado direito. Cortar o pelo, usar dissecando tesoura, ao longo do lado esquerdo do mouse, sobre no meio do caminho entre a frente e pernas traseiras.</li>
	<li>Usando a pinça, pegue o peritônio e cuidadosamente fazer uma incisão para não danificar o baço. Remover o baço com pinça e coloque em um tubo estéril 50 mL contendo cerca de 10 mL de meio de cultura no gelo. Repita para os restantes animais. Nota: O baço é a cor de um grão de feijão, e é mais longo e mais plana do que o rim.</li>
	<li>
Transferi um baço com 2 mL de meio de cultura para uma pequena placa de Petri estéril.
	<ol>
<li>Homogeneizar o baço por moagem é entre a parte geada de dois slides foscas estéril. Se possível, manter a questão e células no meio durante o processo de homogeneização.</li>
		<li>Uma vez completamente homogeneizado, pipeta de 2 mL de meio de cultura contendo os splenocytes através de um filtro de célula de nylon µm 40 em um tubo de 50 mL.</li>
		<li>Repita as etapas acima para os restantes baços usando novos pares de slides geados, tubos de 50 mL com meio de cultura e filtros de célula.</li>
	</ol>
</li>
	<li>Adicione 8 mL de meio de cultura de frio a cada um dos tubos de 50 mL contendo os splenocytes para um volume total de 10 mL/tubo. Determine o número de células por mililitro, usando um hemocytometer.</li>
	<li>Adicione aproximadamente 1 x 106 células/poço para placas 6-poço de cultura. Adicione células ao número de poços que correspondem ao número de diferentes PAMPs sendo usado para o experimento e incluir um controle bem. Adicione 3 mL de meio de cultura ou 3 mL de meio de desafio aos respectivos poços.</li>
	<li>Incube as placas a 37 ° C em 5% de CO2 por 3 h.</li>
	<li>Raspe as células da parte inferior do bem, usando a ponta da pipeta 1.000 µ l anexado a uma micropipeta P1000. Transferi a mídia que contém as células com a mesma ponta da pipeta µ l 1.000 para um tubo de 15 mL.</li>
	<li>Granule as células através de centrifugação a 167 x g durante 5 min à temperatura ambiente. Remover o sobrenadante e lavar o centrifugado com 5 mL de PBS.</li>
	<li>As células de pelotas uma segunda vez a 167 x g por 5 min, retire o sobrenadante e adicionar 600 µ l de tampão de Lise para o centrifugado para lisar as células. Em seguida proceda ao isolamento de RNA.</li>
</ol>4. RNA isolamento e síntese de cDNA
<ol><li>Isolar o RNA do splenocytes usando o kit de extração de RNA de acordo com as instruções do fabricante e realizar a digestão de DNA na coluna 'opcional'.</li>
	<li>Antes da síntese do cDNA, determine a concentração de RNA usando um espectrofotômetro microvolume para verificar se a concentração está dentro da faixa ideal de RNA (até 2 µ g) do kit de síntese do cDNA.</li>
	<li>
Synthesize cDNA para cada uma das amostras usando o kit de transcrição reversa de acordo com as instruções do fabricante. Use 10 µ l de RNA para cada uma das amostras em um volume de reação total de 20 µ l. Após a conclusão da transcrição reversa (thermocycler executado) adicionar 20 μL H2O para cada reação.
</li>
	<li>Construa a curva padrão usando uma micropipeta P10 ou P20 para piscina mRNA de algumas amostras de controlo (por exemplo, 5 µ l de 2 amostras num total de 10 µ l) em um tubo de 0,5 mL para preparar o cDNA.<ol>
<li>Adicione 10 µ l 2 x mestre mistura transcriptase reversa.</li>
		<li>Após a conclusão da transcrição reversa, adicione 10 µ l de H2O para o tubo de reação, que servirá como a concentração inicial ("1") da série de diluição para a curva padrão.</li>
		<li>Execute uma série de diluições de 10 vezes (de 1 a 10-4) adicionando 45 µ l de água utilizando uma micropipeta P100 ou P200 em quatro tubos de 0,5 mL, designados de 10-1, 10-2, 10-3e 10-4.<ol>
<li>Adicionar 5 µ l da concentração inicial "1" no primeiro tubo (10-1) utilizando um micropepitador P20, mistura pipetando para cima e para baixo várias vezes, então transferência 5 µ l do tubo de 10-1 para o tubo designado 10-2 e misture.</li>
			<li>Transferência de 5 µ l utilizando uma micropipeta P20 do tubo de 10-2 no tubo designado 10-3 e misturar. Transferência de 5 µ l utilizando um micropepitador P20 do tubo de 10-3 no tubo designado 10-4 e misture.</li>
		</ol>
</li>
	</ol>
</li>
</ol>5. quantitativo reação em cadeia da polimerase (qPCR)
<ol><li>Determine a quantificação relativa dos níveis de mRNA por qPCR. Dentro do conjunto experimental acima, selecione "Quantificação – curva padrão relativo" e "TaqMan" química. Alterar o volume de reação de 10 µ l. Insira as informações relevantes para o layout da placa (por exemplo, gene alvo, curva de calibração, repórter, etc.).</li>
	<li>Preparar a reação, para que ele contém 0,5 µ l do ensaio da primeira demão/sonda, 5 µ l de mistura mestre de expressão de gene, 2 µ l de H2O e 2,5 µ l de cDNA (10-100 ng).<ol>
<li>Preparar uma mistura de mestre multiplicando cada um dos reagentes anteriores (exceto o cDNA) pelo número de reações, certificando-se de incluir aqueles da curva padrão, controles negativos, realizando cada reação em duplicata e 2 reações extras que serão responsáveis para variação de pipetagem.</li>
		<li>Pipeta de 7,5 µ l da mistura preparada mestre em poços pré-determinado em uma placa de 96 poços de reação com uma micropipeta ou uma pipeta multicanal. Pipetar 2,5 µ l de cDNA no bem apropriado para as incógnitas e os padrões e 2,5 µ l de H2O para os poços de controle negativo.</li>
		<li>Incluir ensaios de primer/sonda para vários genes do relógio molecular e também incluem um ensaio para um controle endógeno (por exemplo, β-actina).</li>
	</ol>
</li>
	<li>A fim de determinar os valores de expressão relativa, calcular a quantidade relativa média para cada replicar, em seguida, para cada amostra, dividir a quantidade relativa média para o gene alvo pela quantidade relativa média para β-actina.</li>
</ol>6. análise estatística
<ol><li>Usando o software de análise estatística, inserir dados no programa sob a opção de estatísticas de coluna. Selecione uma ANOVA One-Way com teste post hoc de Dunnett para avaliar as diferenças entre os valores médios da expressão do gene relógio após desafio PAMP contra o controle.</li>
</ol>

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Neste protocolo, um espectrofotômetro microvolume pode ser usado para quantificar e avaliar a pureza do RNA sendo usado para determinar a expressão do gene. Ácidos nucleicos absorvem UV luz em 260 nm, proteínas normalmente absorvem luz em 280 nm, enquanto outros contaminantes potenciais usados durante um procedimento de extração de RNA (por exemplo, fenol) são detectáveis em 230 nm. Portanto, avaliando a absorvância (A) relação em 260/280 nm (RNA à proteína) e 260/230 nm (RNA de contaminantes não p...

O pulso de disparo mestre em mamíferos, que coordena as oscilações de 24h para quase todos os aspectos da fisiologia e comportamento, está localizado dentro do núcleo supraquiasmático (SCN) do hipotálamo1,2. Além de processos biológicos a nível de regulação, o pulso de disparo mestre também sincroniza periféricos relógios celulares em todo o corpo,3,4,5. Enquanto as máquinas de relógio molecular consiste de pelo menos três loops de feedback transcriptional-translacional interligadas, o núcleo é composto do período (Per1-3), prevenção (Cry1-2), Bmal1, e relógio genes6,7. Além de manter a altura exata do relógio molecular núcleo, alguns produtos de genes do relógio auxiliares (por exemplo, Rev-erbα e Dbp) também regulam a expressão de genes não-tempo, ou seja, clock genes controlados (CCGs)6 , 7.
Funcionais relógios moleculares têm sido descritos em vários tecidos imune (por exemplo, baço e linfonodos)8 e células (por exemplo, B células, as células dendríticas, macrófagos)8,9. Estas células detectam e respondem a padrões moleculares associados patógeno (PAMPs), componentes microbianos conservadas, via receptores de reconhecimento imune inata como receptores Toll-like (TLRs)10. Até à data, têm sido descritos 13 TLRs funcionais, que reconhecem constituintes microbianos, como componentes da parede celular bacteriana, flagelar proteínas e ácidos nucleicos microbial10. O PAMP, lipopolissacarídeo (LPS), um componente da parede celular de bactérias Gram-negativas, reconhecido por TLR4, foi mostrado para alterar o ritmo circadiano nos níveis de organismos e moleculares. Por exemplo, na vivo desafio de LPS induzido atrasos fótica-como fase medido pela atividade em ratos11 e levou à expressão do gene relógio reduzida no SCN e no fígado, conforme determinado pela hibridação in situ e PCR quantitativo, respectivamente, em ratos,12. Depois de um desafio na vivo com LPS, análise de leucócitos do sangue periférico humano13 e tecido adiposo subcutâneo14 revelou expressão alterada de vários genes de relógio, medido através de qPCR. Por último, ex vivo desafios LPS de macrófagos humanos e macrófagos peritoneais de rato, também levados a alteraram a expressão relógio medida pelo qPCR14.
Aqui, descrevemos um protocolo para avaliar a influência dos PAMPs LPS, ODN1826 (oligonucleotídeos sintéticos contendo unmethylated motivos CpG) e matou o calor Listeria monocytogenes (HKLM), reconhecido por TLR4 e TLR9 TLR2, respectivamente, na expressão gênica de relógio molecular em splenocytes de rato. O protocolo inclui esplenectomia rato, isolamento de splenocyte e desafio, RNA extração, síntese do cDNA e qPCR para avaliar a expressão de vários genes do relógio. Este protocolo permite a aquisição atempada de um grande número de células do sistema imunológico com muito pouco animal ou manipulação celular, que pode então ser desafiado ex vivo com diversos PAMPs. O relógio molecular tem sido mostrado para modular a vários aspectos da resposta imune8,15,16, portanto, interrupção do relógio molecular provavelmente iria prejudicar a adequada variação dependente do tempo da resposta imune. Além disso, uma vez que as perturbações do ritmo circadiano podem levar a graves patologias17,18,19,20, pode ser de interesse para os investigadores desafiar splenocytes com uma ampla gama de moléculas e avaliar sua influência sobre o relógio.

<table><tbody><tr><td>Frosted slides</td><td>Fisher</td><td>12-550-343</td><td></td></tr><tr><td>Cell strainers</td><td>Fisher</td><td>22363547</td><td></td></tr><tr><td>Lipopolysaccharide&amp;nbsp;</td><td>InvivoGen</td><td>ltrl-eklps</td><td></td></tr><tr><td>ODN1826</td><td>InvivoGen</td><td>Tlrl-1826-1</td><td></td></tr><tr><td>HKLM</td><td>InvivoGen</td><td>Tlrl-hklm</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>20012-043</td><td></td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104 or 74106</td><td></td></tr><tr><td>RNase-Free DNase Set</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>6-well cell culture plate</td><td>Denville</td><td>T1006</td><td></td></tr><tr><td>50 mL tubes</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>15 mL tubes</td><td>Corning</td><td>352097</td><td></td></tr><tr><td>High Capacity cDNA Reverse Transcription Kit</td><td>ThermoFisher</td><td>4368814</td><td></td></tr><tr><td>TaqMan Gene Expression Assays b-actin</td><td>ThermoFisher</td><td>Mm00607939_s1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Per2</td><td>ThermoFisher</td><td>Mm00478113_m1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Rev-erba</td><td>ThermoFisher</td><td>Mm00520708_m1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Bmal1</td><td>ThermoFisher</td><td>Mm00500226_m1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Dbp</td><td>ThermoFisher</td><td>Mm00497539_m1</td><td></td></tr><tr><td>qPCR machine StepOnePlus</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>ThermoFisher</td><td>4369016</td><td></td></tr><tr><td>MicroAmp Fast 96-well reaction plate (0.1 mL)</td><td>ThermoFisher</td><td>4346907</td><td></td></tr><tr><td>Statistical Analysis Software</td><td>Prism 7.0a</td><td></td><td></td></tr></tbody></table>

the use of mouse splenocytes to assess pathogen-associated molecular pattern influence on clock gene expression

De comportamento de expressão gênica, ritmos circadianos regulam quase todos os aspectos da fisiologia. Aqui, apresentamos uma metodologia para desafiar splenocytes de rato com o lipopolissacarídeo (PAMPs) de padrões moleculares associados patógeno (LPS), ODN1826 e calor-matou Listeria monocytogenes e examinar seu efeito sobre o molecular circadian pulso de disparo. Anteriormente, estudos têm focado a analisar a influência dos LPS do relógio molecular, usando uma variedade de abordagens in vivo e ex vivo de uma variedade de modelos (por exemplo, rato, rato e humanos). Este protocolo descreve a isolação e o desafio de splenocytes, bem como a metodologia para avaliar a expressão de gene de relógio pós-desafio através de PCR quantitativo. Esta abordagem pode ser usada para avaliar não só a influência de componentes microbianos do relógio molecular mas também outras moléculas que podem alterar a expressão do relógio. Esta abordagem poderia ser utilizada para arreliar distante o mecanismo molecular de como interação de PAMP-Toll-like receptor influencia a expressão do relógio.

Os ratos foram sacrificados em ZT13, splenocytes foram isoladas e desafiou ex vivo com o LPS PAMPs, ODN1826 ou HKLM. Depois de 3 h, RNA foi isolado, e qPCR foi usada para avaliar os níveis de expressão relativa dos genes relógio molecular relógio, Per2, Dbp e Rev-erbα em comparação com células de controle sem contestação. Após o desafio PAMP, níveis de expressão de relógio não foram significativamente diferentes do que a...

Watch this Scientific Journal Video about The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression at JoVE.com

The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

Este protocolo descreve uma técnica usando splenocytes rato para descobrir padrões moleculares associados patógeno que alteram a expressão de gene do relógio molecular.

O uso do Mouse Splenocytes para avaliar a influência do patógeno associado padrão Molecular na expressão gênica de relógio

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse ...

the-use-mouse-splenocytes-to-assess-pathogen-associated-molecular

Universidad San Sebastian

Research

JoVE Journal

Genetics

7.4K visualizações.  University of Hartford. Este protocolo descreve uma técnica usando splenocytes rato para descobrir padrões moleculares associados patógeno que alteram a expressão de gene do relógio molecular.

Vídeo: The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Medição paralela da expressão do gene relógio circadiano e hormonal secreção em culturas de células primárias humanas

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Genética

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

An in vitro bioluminescence assay to determine cellular circadian rhythm in mammary epithelial cells is presented. This method utilizes mammalian cell reporter plasmids expressing destabilized luciferase under the control of the PERIOD 2 gene promoter. It can be adapted to other cell types to evaluate organ-specific effects on circadian rhythm.

In Vitro Ensaio de bioluminescência para caracterizar o ritmo circadiano em células epiteliais mamárias

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

Ordenação temporal de Expressão Dynamic Data de expressão espacial mapas detalhados

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Biologia do Desenvolvimento

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

Medição quantitativa da resposta imune e do sono em<em&gt; Drosophila</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Imunologia e Infecção

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

A seguir em tempo real o impacto de fatores de virulência do pneumococo em um mouse Pneumonia aguda Modelo Usando bactérias bioluminescentes

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Um modelo de rato para induzida por patógeno inflamação crônica no local e sistêmica Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal allophycocyanin (APC) fluorescence intensity, are conjugated to antibodies specific to a particular analyte. Next, a selected panel of defined capture bead sets is incubated with a biological sample containing target analytes specific to the capture antibodies. A biotinylated detection antibody cocktail is added, which leads to the formation of capture bead-analyte-detection antibody sandwiches.
Finally, streptavidin-phycoerythrin (SA-PE) is added, which binds to biotinylated detection antibodies, providing fluorescent signal intensities in proportion to the amount of bound analyte. The PE fluorescent signal of analyte-specific beads regions is quantified using flow cytometry, and the concentrations of particular analytes are determined using data analysis software and the standard curve generated in the assay.
In this experiment, we use a mouse T helper cytokine panel to simultaneously quantify the concentration of 13 separate cytokine targets in tissue culture supernatants collected from mouse splenocytes cultured under various stimulatory conditions.

This protocol describes the quantification of multiple cytokine targets simultaneously in tissue culture supernatants collected from stimulated mouse splenocytes using multiplex bead based immunoassay platform and a flow cytometer.

Multiplexar Cytokine perfilação de Mouse estimulada Splenocytes usando uma plataforma baseada em Cytometric Bead imunoensaio

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

Usando um patógeno bacteriano para sonda para celular e organísmica-nível Host respostas

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Avaliação das respostas do hospedeiro-patógeno e eficácia da vacina em camundongos

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

Imagem latente da produção in situ da gama do interferon no spleen do rato depois da infecção dos monocytogenes de Listeria

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...