The authors thank the Central Animal facility of the University Hospital Aachen for technical support. This work was supported by the German Research Foundation (DFG Ta434/2-1, DFG SFB/TRR 57) and by the Interdisciplinary Center for Clinical Research (IZKF) Aachen. This work was further supported by the Core Facility &#8221;Two-Photon Imaging&#8221;, a Core Facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH Aachen University.

NOTA: Os experimentos foram realizados em conformidade com a legislação alemã que rege os estudos em animais após o &#39;Guia para o cuidado e uso de animais de laboratório &quot;(publicação NIH, 8ª edição, 2011) e da Directiva 2010/63 / UE relativa à protecção dos animais utilizados para fins científicos (Jornal Oficial da União Europeia, 2010). Permissão oficial foi concedida a partir do cuidado com os animais e uso de escritório governamental (LANUV Nordrhein-Westfalen, Recklinghausen, Alemanha). Nota: Os passos que podem ser omitidos para a imagem latente de curto prazo (por exemplo, tirar fotos, pilhas 3-D ou também de microscopia de lapso de tempo curta duração) são marcados com asterisco (*) para reduzir o tempo de preparação e simplificar o protocolo cirúrgico. Imagiologia também pode ser realizada sem grande controlo e de circulação, se necessário, no entanto, o tempo de sobrevivência será acentuadamente reduzido. 1. Setup Microscópio e pré-cirurgia Preparação (5-10 min) <ol><li> Sistema Ligue (microscópio, laboratório de energia, laser, almofada de aquecimento, câmara climática, web cam, dispositivo de bomba de seringa, respirador). </li><li> Ligue O 2 de abastecimento para Isoflurane Vapor e definir o nível de isoflurano para 1,5 Vol% (v / v), conectar-se a um pequeno respirador animal. <ol><li> Para imagens de curto prazo, manter a anestesia com isoflurano somente após a indução inicial anestesia ip (veja o passo 2.2). Em seguida, use 2 Vol% (v / v) isoflurano, e manter o tempo de imagem inferior a 2 horas para garantir estável microcirculação hepática. </li></ol></li><li> Definir a respiração 120-140 ciclos respiratórios / min com um volume de 150-200 uL. </li><li> Configure fase rato agarose. Preparar 100 ml de 3% (w / v) de agarose, vertendo-a em um prato (aproximadamente 10 cm x 15 cm base) num ângulo de 40 °. </li><li> Configure área de trabalho cirúrgico coletando a instrumentação necessária. Para prevenir a infecção microbiana, desinfectar instrumentos usando uma solução a 1% de Sekusept Forte S (9,9% w / v), Glutaral 9,8% w / v, formaldeído durante 5 minutos antes da experiência (Figura 1A). </li><li> Obter restantes componentes: desinfectante da pele (por exemplo, solução de povidona iodo de 10%), no fígado (fase de pilhas e ponte), solução de agarose (3% (w v) em PBS /), bomba de seringa com 5% (w / v) de uma solução de glucose (G-5), bomba de seringa com solução anestésica I (cetamina 0,1 mg / ml, xilazina 0,5 mg / ml, 5 ug de fentanil / ml), cotonetes, n-Butil-2-Cyanoacrylat, e 1x PBS. Coloque agarose fase prato em câmara de incubação para o pré-aquecimento. </li></ol> 2. A traqueotomia (10-15 min) <ol><li> Use camundongos C57BL / 6 a partir da casa de reprodutoras de 25-28 g. Casa os ratinhos sob condições específicas isentas de patogénios de acordo com as orientações FELASA, num ambiente de temperatura e humidade controladas com um ciclo luz / escuro de 12 h de 12 horas de luz. Permitir que os animais livre acesso à dieta padrão ratos (Sniff Padrão Rodent Diet) e água ad libitum. </li><li> Anestesiar o mouse pelo initial intraperitoneal (ip) de injecção de 250 ul de solução de anestésico cetamina (II 0,1 mg / ml, xilazina 1 mg / ml, de Buprenorfina 10 ug / ml). <ol><li> * Para manutenção durante imagens intravital, administrar continuamente solução anestésica I por injecção contínua ip (cetamina 0,1 mg / ml, xilazina 0,5 mg / ml, 5 ug de fentanil / ml) usando um dispositivo de bomba de seringa com uma taxa de fluxo de 0,2 ml / h em combinação com inalação de isoflurano 0,5 vol% (v / v). </li><li> Verifique reflexos após 5 min (por exemplo pitada almofada do pé com uma pinça), desinfectar a pele de traqueostomia, preparação começar se o mouse está em estado de anestesia cirúrgica tolerante. </li><li> Aplicar olho creme de protecção (por exemplo, dexpantenol 50 mg / g) para evitar a córnea de secar (Figura 1B). </li></ol></li><li> Fixar do mouse sobre a preparação mesa expondo lado ventral usando fita adesiva para as extremidades; overstretch suavemente pescoço, fixar nessa posição, por exemplo., através da utilização de uma faixa de borracha t enganchadoo incisivos. <ol><li> Desinfectar pele e da pele utilizando uma solução de povidona-iodo, mediante a aplicação de desinfectante com uma cotonete. </li></ol></li><li> Efetuar corte inicial pele (0,5-1 cm de comprimento) diretamente abaixo do queixo (Figura 1C) <ol><li> Dissecar cuidadosamente tecido conjuntivo entre as glândulas salivares (Figura 1D). </li><li> Retire com cuidado aberta muscular tubo circundante traquéia (Figura 1E, F). </li></ol></li><li> Coloque fio cirúrgico debaixo da traquéia para posterior fixação do tubo de ventilação (Figura 1G). <ol><li> Abrir traqueia com micro-tesoura entre anéis cartilaginosos realizando uma incisão em forma de T (Figura 1H) </li></ol></li><li> Coloque tubo de ventilação na traquéia através da incisão (~ 0,5 centímetros). Para empurrar o tubo para a frente, pegue final caudal com pequenas pinças anatômicas e avançar gentilmente o tubo na traquéia (Figura 1-I) </li><li> Fixar tubo com fio cirúrgico (por exemplo, </em&gt;, 5-0) com a linha colocada no Passo 2.5 dando um nó em torno de tubo no interior da traquéia; executar segunda fixação cranial do tubo na pele para evitar depositioning acidental (Figura 1J, K). </li><li> Corte do selo com cola de tecidos (por exemplo, n-butil-2-Cyanoacrylat) (Figura 1D). </li><li> Fixar tubo na cabeça, usando fita adesiva. </li></ol> 3. laparotomia (15-20 min) <ol><li> Coloque o mouse sobre almofada de aquecimento para evitar a hipotermia. Shave abdômen, retire cuidadosamente o cabelo da pele. Desinfetar pele raspada e pele com a solução de iodopovidona. </li><li> Realize uma pequena pele cortada abaixo do esterno utilizando uma tesoura cirúrgica (Figura 2A). Estender o corte lateralmente abaixo das costelas para ambos os lados, cauterização todos os vasos sanguíneos visíveis para evitar sangramento. </li><li> Executar cuidadosamente um pequeno corte na linha alba abaixo do esterno, a abertura do peritônio (Figura 2B). Estender o corte para ambos os lados usando cauterizção para impedir o sangramento (Figura 2C, D). </li><li> Coloque o mouse em agarose fase prato (Figura 2E). Inserir as pilhas de altura adequada em ambos os lados do rato (geralmente 12-14 lamelas) (Figura 2F). </li><li> Coloque sensor de gatilho respiratório debaixo parte superior das costas para sincronizar o microscópio com a respiração. Ative unidade de gatilho (Figura 2G). </li><li> Coloque fio cirúrgico (5-0) através do esterno para retrair costelas (Figura 2?). </li><li> Cuidadosamente corte ligamento ligar fígado e diafragma (ligamento falciforme), bem como do tracto gastrointestinal e fígado usando tesouras cirúrgicas curvas. Cortar o ligamento falciforme para baixo na aorta (Figura 2J). </li></ol> 4. Configuração da amostra (10-15 min) <ol><li> Coloque o mouse sobre o lado direito, com um ângulo de 45 ° para facilitar o acesso a grande lobo hepático. </li><li> Adicionar ponte encenar abaixo das costelas, que abrange o abdominalcavidade. Fabricação de uma ponte, usando uma lâmina de cobertura padrão com revestimento de fita adesiva para cobrir as bordas cortantes (Figura 2K). </li><li> Coloque grande lobo hepático no palco. Deslizar cuidadosamente sonda stylus dupla bola ou uma espátula acolchoado abaixo do fígado e segure a parte superior do órgão com um cotonete molhado ou tecido molhado. Levante lobo para o slide e aplicar arrastar suave. Lobe pode ser dobrado ou enrolado. Prestar cuidados extra para só manipulação suave do fígado (Figura 2L). </li><li> Coloque estacas laterais de apoio, por exemplo, pilhas de pequenas peças de cobertura (20 mm x 20 mm), com aproximadamente a mesma altura do lóbulo do fígado ao lado dela para suportar a cobertura de deslizamento. </li><li> Coloque grande lamela (24 mm x 50 mm) no lóbulo do fígado. Certifique-se de que lamela é orientada mais horizontal possível (Figura 2 M). NOTA: A cobertura de vidro deve estar em contato com o tecido sem apertar. Verifique se há sinais visíveis de fluxo sanguíneo prejudicado (tecido branco). Se microcircmento é interrompido, adicionar um maior apoio stakes. </li><li> * Coloque dois cateteres intraperitoneal independentes (Figura 2 N) para anestesia longo prazo e G-5 aplicação. Instale cateteres lateralmente na parte inferior do abdômen ao lado dos membros posteriores. NOTA: Os cateteres intraperitoneais podem ser auto-fabricados através da ligação 27 agulhas G com tubagem de silicone flexível (Figura 3). </li><li> * Agulha Fixate com um laço de 5-0 cirúrgica na pele para evitar o deslocamento acidental. </li><li> * Prenda bomba de seringa com solução anestesia I à cateterização 1, defina a taxa de fluxo de 0,2 ml / h; anexar bomba de seringa com G-5 ao cateter 2, definir taxa de fluxo de 0,1 ml / hr. </li></ol> 5. Rato Monitoring <ol><li> * Coloque eletrodos de ECG em frente e as extremidades traseiras (Figura 4A). </li><li> * Conecte a tubulação de expiração para o CO 2 sensor de nível. </li><li> Adicionar sensor de temperatura externo conectado a almofada de calor (Figura 4C). </li></ol> 6. Incorporação e fixação do tecido (5-10 min) <ol><li> Preparar 100 ml de 3% (w / v) de agarose em 1X PBS. Incorporar fígado quando a temperatura é 41 ° C, utilizando uma seringa de 5 ml e uma agulha de 18 G (Figura 5A). </li><li> Pour agarose restante em lamela e em torno do rato (Figura 5B, C). Espere até agarose está totalmente gelatinizado. </li><li> Retire o excesso de agarose usando a espátula Heidemann, preparando uma grande viewfield suficiente para digitalizar o lobo hepático preparado (Figura 5D, E). </li></ol> 7. Criação de Imagens <ol><li> Transferir o mouse na câmara de microscópio climáticas. </li><li> Adicionar 50-100 ml de 1x PBS (pré-aquecido a 37 ° C). </li><li> Cubra amostra prato para evitar a evaporação (Figura 5F). </li><li> * Diminuição isoflurano inalativa de 0,5 Vol% (v / v). </li><li> * Iniciar o monitoramento de software, a gravação de ECG, freqüência cardíaca e expiratória CO 2. </li><li> Comece imagem: abrir lapersianas SER, identificar campos exibição de interesse, definir limites superior e inferior para Z-stacks, e começar a tempo de gravação lapso. Reajustar Z pilhas se necessário, para corrigir-Z deriva (ex., Devido a alterações na pressão sanguínea ou a variação de temperatura, Figura (5G). NOTA: Após a conclusão do período de imagem, ou se a circulação ou anestesia dos animais torna-se instável, sacrificar os ratinhos por deslocamento cervical (sem despertar da anestesia). </li></ol>

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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+recruitment+of+the+inflammatory+Gr1++monocyte+subset+upon+liver+injury+promotes+hepatic+fibrosis.">Hepatic recruitment of the inflammatory Gr1+ monocyte subset upon liver injury promotes hepatic fibrosis.</a> Hepatology. 50 (1), 261-274 (2009).</li><li>Heymann, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+macrophage+migration+and+differentiation+critical+for+liver+fibrosis+is+mediated+by+the+chemokine+receptor+C-C+motif+chemokine+receptor+8+in+mice.">Hepatic macrophage migration and differentiation critical for liver fibrosis is mediated by the chemokine receptor C-C motif chemokine receptor 8 in mice.</a> Hepatology. 55 (3), 898-909 (2012).</li><li>Ramachandran, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Ly-6C+expression+identifies+the+recruited+macrophage+phenotype,+which+orchestrates+the+regression+of+murine+liver+fibrosis.">Differential Ly-6C expression identifies the recruited macrophage phenotype, which orchestrates the regression of murine liver fibrosis.</a> Proc Natl Acad Sci U S A. 109 (46), E3186-E3195 (2012).</li><li>Moles, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+TLR2/S100A9/CXCL-2+signaling+network+is+necessary+for+neutrophil+recruitment+in+acute+and+chronic+liver+injury+in+the+mouse.">A TLR2/S100A9/CXCL-2 signaling network is necessary for neutrophil recruitment in acute and chronic liver injury in the mouse.</a> J Hepatol. 60 (4), 782-791 (2014).</li><li>Hammerich, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine+receptor+CCR6-dependent+accumulation+of+&#947;&#948;+T+cells+in+injured+liver+restricts+hepatic+inflammation+and+fibrosis.">Chemokine receptor CCR6-dependent accumulation of &#947;&#948; T cells in injured liver restricts hepatic inflammation and fibrosis.</a> Hepatology. 59 (2), 630-642 (2014).</li><li>Syn, W. -. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKT-associated+hedgehog+and+osteopontin+drive+fibrogenesis+in+non-alcoholic+fatty+liver+disease.">NKT-associated hedgehog and osteopontin drive fibrogenesis in non-alcoholic fatty liver disease.</a> Gut. 61 (9), 1323-1329 (2012).</li><li>McDonald, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+CD44+and+hyaluronan+is+the+dominant+mechanism+for+neutrophil+sequestration+in+inflamed+liver+sinusoids.">Interaction of CD44 and hyaluronan is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids.</a> J Exp Med. 205 (4), 915-927 (2008).</li><li>Egen, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+reveals+limited+antigen+presentation+and+T+cell+effector+function+in+mycobacterial+granulomas.">Intravital imaging reveals limited antigen presentation and T cell effector function in mycobacterial granulomas.</a> Immunity. 34 (5), 807-819 (2011).</li><li>Singer, G., Stokes, K. Y., Granger, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+microcirculation+in+murine+sepsis:+role+of+lymphocytes.">Hepatic microcirculation in murine sepsis: role of lymphocytes.</a> Pediatr Surg Int. 24 (1), 13-20 (2008).</li><li>Phillipson, M., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neutrophil+in+vascular+inflammation.">The neutrophil in vascular inflammation.</a> Nat Med. 17 (11), 1381-1390 (2011).</li><li>Khandoga, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+and+quantitative+analysis+of+leukocyte+directional+migration+and+polarization+in+inflamed+tissue.">In vivo imaging and quantitative analysis of leukocyte directional migration and polarization in inflamed tissue.</a> PLoS One. 4 (3), e4693 (2009).</li></ol>

The authors disclose no con&#64258;ict of interests.

O objetivo do nosso estudo foi desenvolver um método altamente padronizada, estável e reprodutível para a imagem latente TPLSM intravital do fígado. Imaging Intravital em geral deu informações valiosas sobre o comportamento celular sob condições reais seguintes homing e interação de diferentes populações de leucócitos em desenvolvimento, a homeostase e da doença. No entanto, a posição anatómica um pouco difícil de fígado, devido a que movimento respiratório e intestinal peristáltica são transmitido...

A visualização de células e as funções celulares em órgãos inteiros ou até mesmo de organismos inteiros tem sido de grande interesse para mais de 50 anos, incluindo praticamente todas as partes do corpo 2. Por isso, alguns estudos iniciais já empregaram imagens intravital do fígado 3,4. No entanto, existem várias limitações atualizado sobre estável imagens de alta resolução a longo prazo do tecido do fígado. Devido à posição anatómica do fígado em estreito contacto com o diafragma e o tracto gastrointestinal 5, o problema mais comum para imagiologia intravital microscópica é o movimento devido à respiração e, em menor grau, peristáltica do tracto intestinal 6. Em comparação com outros órgãos sólidos, cirurgia do fígado é particularmente desafiador. Devido à estrutura microvascular densa, manipulação cirúrgica pode conduzir a lesões hemorrágicas maciças, microcirculação comprometida 7 e também a activação de residente immune células, tais como células de Kupffer 8. Portanto, a fixação mecânica do tecido como publicado anteriormente 6,9 é susceptível de interferir com a imagem de microscopia intravital. Em um fígado saudável, 10-15% do volume total de sangue reside dentro da vasculatura hepática, e o órgão recebe cerca de 25% do débito cardíaco geral 10, tornando o órgão altamente susceptíveis a mudanças na circulação (por exemplo, flutuações de pressão arterial ). Portanto, as interrupções no fluxo sanguíneo hepático devido, por exemplo, tensão de cisalhamento, deslocamento, lesão por manuseio excessivo dos tecidos ou circulação centralizada vai levar a alterações artificiais no comportamento migratório de leucócitos, deficiente oxigenação fígado e, portanto, mais danos ao fígado, afetando as respostas imunes do fígado, bem como preservação de órgãos e tempo de vida total do animal. Os primeiros estudos microscópicos foram baseados em mi epifluorescência intravitalcroscopy, mas várias restrições técnicas, tais como o branqueamento foto e baixa profundidade de penetração limitar o uso desta técnica para imagiologia do fígado a longo prazo 4,11,12. Com o desenvolvimento da microscopia multiphoton na década de 1990, as limitações de branqueamento foto ou profundidade de penetração foram principalmente resolvido, já que este novo método era tecnicamente capaz de realizar estudos de imagem em praticamente todos os órgãos em situações da vida real 13-15. No entanto, os principais desafios que subsistem no que diz respeito à geração de imagens de fígado foram: movimentos de respiração, autofluorescência do tecido do fígado, protegendo o fluxo sanguíneo inalterado nos sinusóides hepáticos, e de imagem especialmente estáveis ​​por longos períodos de várias horas 16. Apesar de vários estudos abordaram a função e migração de vários leucócitos no fígado 17, por exemplo, a NKT células-18-20, células T 21,22, os macrófagos do fígado 23,24 ou neutrófilos 25, a longo prazo multiphoton mimaging icroscopy ainda não tinha sido estabelecida com sucesso, uma tarefa ainda mais difícil em animais com doença hepática aguda ou crônica devido ao dano existente e, portanto, maior susceptibilidade a danos maiores 26. No entanto, monitorando o comportamento migratório e função celular de leucócitos no fígado em tempo real, permite que novas idéias em seu papel especial na homeostase do fígado e doença 27. O receptor de quimiocina CXCR6 é expresso em vários subconjuntos de linfócitos, incluindo células assassinas naturais (NK), as células NKT e algumas populações de células T 18,28. Estudos anteriores em ratos indicaram que CXCR6 e sua CXCL16 ligando cognato pode controlar o patrulhamento de células NKT em sinusóides hepáticos durante a homeostase. Por conseguinte, a utilização de ratinhos CXCR6.gfp (transportando um knock-in de proteína fluorescente verde [GFP] no locus CXCR6) tem sido descrita para investigar a migração de linfócitos em vários órgãos tais como o cérebro 29e também fígado 18,20, mostrando aumento da infiltração de células CXCR6.gfp sobre a inflamação. Com o método proporcionado no presente estudo foi possível seguir estes processos durante um longo período de tempo sob condições estabilizadas. O procedimento baseado multiphoton intravital permitido de imagem que foi altamente reprodutível com perturbação mínima do animal e do órgão; otimizado para a sobrevivência dos animais a longo prazo através de monitoramento extensa, seguida de perto o controle da respiração e circulação; e altamente flexível e fácil de adoptar igualmente para outros órgãos parenquimatosos, tais como de rim ou baço.

<table border="0" cellpadding="0" cellspacing="0" width="1184">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Anesthetics</td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td style="width:293px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Buprenorphine</td>
			<td style="width:263px;">Essex Pharma</td>
			<td style="width:219px;">997.00.00</td>
			<td>Analgeticum, 0.1 mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fentanyl</td>
			<td style="width:263px;">Rotex Medica</td>
			<td style="width:219px;">charge: 30819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fluovac anesthesia system</td>
			<td style="width:263px;">Harvard Apparatus</td>
			<td style="width:219px;">34-1030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Glucose 5%</td>
			<td style="width:263px;">Braun</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ISOFLO (Isoflurane Vapor) vaporiser</td>
			<td style="width:263px;">Eickemeyer</td>
			<td style="width:219px;">4802885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Isoflurane</td>
			<td style="width:263px;">Forene Abbott</td>
			<td style="width:219px;">B 506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Isotonic (0.9%) NaCl solution</td>
			<td style="width:263px;">DeltaSelect GmbH</td>
			<td style="width:219px;">PZN 00765145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Ketamin 10%</td>
			<td style="width:263px;">ceva</td>
			<td style="width:219px;">Charge: 36217/09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Xylazin 2%</td>
			<td style="width:263px;">medistar</td>
			<td style="width:219px;">Charge: 04-03-9338/23</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Consumable supplies</td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">20ml Syringe</td>
			<td style="width:263px;">BD Plastipak</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250ml Erlenmeyer flask</td>
			<td style="width:263px;">Schott Duran</td>
			<td>21 226 36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">25mL Beaker 2x</td>
			<td style="width:263px;">Schott Duran</td>
			<td style="width:219px;">50-1150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">2ml syringe</td>
			<td style="width:263px;">BD Plastipak</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-0 Vicryl suture</td>
			<td style="width:263px;">Ethicon</td>
			<td style="width:219px;">V7980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Agarose</td>
			<td style="width:263px;">commercially available</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Bepanthen Eye and Nose ointment</td>
			<td style="width:263px;">Bayer Vital GmbH</td>
			<td style="width:219px;">6029009.00.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Change-A-Tip Deluxe High-Temp Cautery Kit</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">18010-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Cotton Gauze swabs</td>
			<td style="width:263px;">Fuhrmann GmbH</td>
			<td style="width:219px;">32014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Cover Slip 24x50mm</td>
			<td style="width:263px;">ROTH</td>
			<td style="width:219px;">1871</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Durapore silk tape</td>
			<td style="width:263px;">3M</td>
			<td style="width:219px;">1538-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Feather disposable scalpel</td>
			<td style="width:263px;">Feather</td>
			<td style="width:219px;">02.001.30.011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fine Bore Polythene Tubing 0,58mm ID</td>
			<td style="width:263px;">Smiths medical</td>
			<td style="width:219px;">800/100/200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Histoacryl</td>
			<td style="width:263px;">Braun</td>
			<td style="width:219px;">1050052</td>
			<td>5x 0,5ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Leukoplast</td>
			<td style="width:263px;">BSN Medical Inc.</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Microscope Slides</td>
			<td style="width:263px;">ROTH</td>
			<td style="width:219px;">1879</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Poly-Alcohol Haut&#8230;farblos Antisepticum</td>
			<td style="width:263px;">Antiseptica GmbH</td>
			<td style="width:219px;">72PAH200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Sterican needle 18 G x 1</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;">304622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Sterican needle 27 3/4 G x 1</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;">4657705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Tissue paper</td>
			<td style="width:263px;">commercially available</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Surgical Instruments</td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amalgam burnisher 3PL</td>
			<td style="width:263px;">Gatz</td>
			<td style="width:219px;">0110?</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blair retractors (4 pronged (blunt)) x2</td>
			<td style="width:263px;">Storz&#38;Klein</td>
			<td style="width:219px;">S-01134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont No.7 forceps</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">91197-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Graefe forceps curved x1</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11151-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Graefe forceps straight x2</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11050-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heidemann spatula HD2</td>
			<td style="width:263px;">Stoma</td>
			<td style="width:219px;">2030.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Needle holder Mathieu</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">12010-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Scissor</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">14074-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Semken forceps</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11008-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Small surgical scissors curved</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">14029-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Small surgical scissors straight</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">14028-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Standard pattern forceps</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11000-12</td>
			<td></td>
		</tr>
		<tr height="21">
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			<td style="width:263px;">Fine Science Tools Inc.</td>
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long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using cxcr6.gfp reporter mice

<html> Inflamação do fígado como uma resposta à lesão é um processo altamente dinâmico envolvendo a infiltração de subtipos distintos de leucócitos incluindo monócitos, neutrófilos, subconjuntos de células T, células B, células assassinas naturais (NK) e as células NKT. Microscopia intravital do fígado para monitorar a migração de células imunes é particularmente desafiador devido às altas exigências em matéria de preparação de amostras e fixação, resolução óptica e sobrevivência dos animais no longo prazo. No entanto, a dinâmica dos processos inflamatórios, assim como estudos de interacção celular poderia fornecer informações importantes para compreender melhor o início, a progressão e regressão da doença inflamatória do fígado. Portanto, um método altamente sensível e confiável foi criada para estudar a migração e célula-célula-interações de células imunes diferentes em fígado de rato durante longos períodos (cerca de 6 h) por dois fótons de microscopia de varredura a laser intravital (TPLSM) em combinação com tratamento intensivo monitorização. ent &quot;&gt; O método fornecido inclui uma preparação suave e fixação estável do fígado com perturbação mínima do órgão; imagiologia intravital longo prazo usando microscopia multiphoton multicolor com praticamente nenhuma fotodegradação ou efeitos fototóxicos, durante um período de até 6 horas, permitindo rastreamento de subconjuntos de leucócitos específicos; e condições de imagem estável devido à ampla fiscalização do rato parâmetros vitais e estabilização da circulação, temperatura e troca gasosa. Para investigar a migração de linfócitos após a inflamação do fígado CXCR6.gfp ratinhos knock-in foram submetidos a imagiologia do fígado intravital em condições basais e após os danos do fígado aguda e crónica induzida por injecção intraperitoneal (s) de tetracloreto de carbono (CCl 4). CXCR6 é um receptor de quimiocina expressa em linfócitos, principalmente em Natural T assassinas (NKT) -, Natural Killer (NK) - e subconjuntos de linfócitos T, tais como células T CD4 +, mas também asso mucosasated invariante células (MAIT) T 1. Seguindo o padrão migratório e posicionamento de CXCR6.gfp + células imunes permitiu uma visão detalhada sobre o seu comportamento alterado após a lesão hepática e, portanto, o seu potencial envolvimento na progressão da doença.

Para validar nossa abordagem TPLSM intravital, nós submetido GFP / + camundongos CXCR6 a imagem TPLSM intravital. Os ratos foram quer deixados sem tratamento como controlos de linha de base ou submetido a uma única injecção intraperitoneal de tetracloreto de carbono (CCl 4) para induzir danos no fígado aguda 20. As sequências de vídeo foram tomadas ao longo de um período de tempo de 2-5 horas, e as células foram traçadas ao longo do tempo, devido à...

Watch this Scientific Journal Video about Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice at JoVE.com

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Long Term Intravital Multiphoton Microscopia de imagem de células imunes no saudáveis ​​e doentes do fígado por meio CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

long-term-intravital-multiphoton-microscopy-imaging-immune-cells

Research

JoVE Journal

Immunology and Infection

11.8K visualizações.  RWTH University-Hospital Aachen. Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Vídeo: Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Sete Passos para Stellate Células

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Imunologia e Infecção

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Imagem intravital da Thymus rato usando 2-Photon Microscopia

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

Crônica de Longo Prazo<em&gt; Pseudomonas aeruginosa</em&gt; Infecção das vias aéreas em ratos

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Altamente Resolvido Microscopia intravital Striped-iluminação dos centros germinativos

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

Microscopia intravital Imagem do Fígado seguinte<em&gt; Leishmania</em&gt; Infecção: Uma Avaliação de hepáticas Hemodinâmica

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

Interações Intravital Microscopia de leucócito-endothelial e plaquetas-leucócitos em mesenterial Veias Ratos

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Análises de<em&gt; Yersinia enterocolitica</em&gt; Effector translocação em células hospedeiras Usando Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Imagiologia neutrófilos e monócitos em Mesenteric Veins por Microscopia intravital em ratos anestesiados em Tempo Real

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

Intravital Imaging of Neutrophil Priming Using IL-1&amp;#946; Promoter-driven DsRed Reporter Mice

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

Imagem intravital de neutrófilos Priming Usando IL-1-driven promotor DsRed Mice Reporter

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Mycobacterium tuberculosis em camundongos com fluorescência de enzima do repórter de imagem

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Long Term Intravital Multiphoton Microscopia de imagem de células imunes no saudáveis ​​e doentes do fígado por meio CXCR6.Gfp Reporter Mice

Long Term Intravital Multiphoton Microscopia de imagem de células imunes no saudáveis e doentes do fígado por meio CXCR6.Gfp Reporter Mice