Este trabalho foi financiado em parte por concessões do National Institutes of Health (P30 HL101302 e RO1 HL109439 a JC) e da American Heart Association (SDG 5.270.005 de JC). A. Barazia foi apoiado por uma NIH T32HL007829 treinamento subvenção.

1. Preparação de microscópio intravital (Figura 1A) <ol><li> Preparar tampão de superfusão (125 mM NaCl, 4,5 mM KCl, 2,5 mM CaCl2, 1 mM de MgCl2, e 17 mM de NaHCO3, pH 7,4). </li><li> Transformar-se num banho de água para manter a temperatura circulatório de tampão e um cobertor de termo-regulado a 37 ° C. Arejar tampão com gás de azoto (5% de CO 2 equilibrada com azoto). </li><li> Ligue o sistema de microscópio (Sutter Lambda DG-4 de alta velocidade de onda changer, trabalho com computador, microscópio Olympus BX61W, MPC-200 de multi-manipulador, o ROE-200 controlador de fase, câmera de alta velocidade, e intensificador). </li><li> Para o modelo de inflamação vascular, usar um espelho dicróico de 100%. Para induzir a formação de trombos por uma lesão laser, substituir um espelho 100% com um espelho de 50/50% e ligar um sistema de ablação por laser Micropoint. </li></ol> 2. Preparação do músculo cremaster para microscopia intravital (Figura 1B) <ol><li> Anestesiar umrato macho (6-8 semanas de idade, ratinhos C57BL / 6) por injecção ip de cetamina (125 mg de peso corporal / kg (PC)) e xilazina (12,5 mg / kg de peso corporal). A Universidade de Illinois Animal Care Institucional e Comitê de Uso aprovado todo o cuidado animal e procedimentos experimentais. Para um modelo de inflamação vascular, injectar murina TNF-α (0,5 ug em 250 uL de solução salina) intrascrotally num rato hr 3 antes de imagiologia (2-2,5 horas antes da cirurgia). </li><li> Colocar o rato sobre um cobertor de termo-regulado a 37 ° C num tabuleiro de microscópio intravital. </li><li> Puxar para cima da pele sobre a superfície do gargalo e com a pinça incisão horizontal da linha média da pele do pescoço até 1 cm com uma tesoura. </li><li> Abra cuidadosamente a musculatura cervical com uma tesoura sem corte e retire o músculo ao redor da traquéia. </li><li> Canular um tubo PE90 na traqueia para eliminar a dificuldade de respiração. </li><li> Remover suavemente o lado esquerdo do músculo do colo do útero e isolar a veia jugular dos tecidos vizinhos. </li><li> C annuum tubo PE10 tarde na veia jugular esquerda para infusão de anestésicos e de anticorpos adicionais. </li><li> Retire cuidadosamente e incisão na pele do escroto horizontalmente. Para manter a integridade do tecido ao longo do experimento, tampão pré-aquecido foi superfundidos sobre o campo cirúrgico. </li><li> Pressione na parte inferior do abdômen com uma pinça e retire cuidadosamente um testículo com a pinça outro. </li><li> Remover os tecidos conjuntivos circundantes em torno dos testículos. </li><li> Faça uma incisão no músculo cremaster verticalmente e achatar o músculo mais de uma lamela de vidro em uma bandeja de microscópio intravital fixando a periferia. </li><li> 10-20 min para permitir que o músculo se estabilizar antes da recolha de dados. Arteríolas do músculo cremaster (para a formação de trombo) e vénulas (por inflamação vascular), com um diâmetro de 30-45 mM foram escolhidos para o nosso estudo. </li><li> Para manter as condições de anestesia, infundir 30-50 ml do mesmo anestésico min aproximadamente a cada 30 através do cannulus jugular. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 3. Microscopia intravital para TNF-α induzida por inflamação vascular <ol><li> Após a colocação do mouse sobre o microscópio intravital, aberto SlideBook software 5.0 e clique em &quot;Janela de foco&quot; na barra de menu para definir a ótica adequadas e objetivas (60X). </li><li> Infundir Dylight 488-conjugado de rato anti-ratinho CD42c (0,1 ug / g BW em 100 ul de solução salina) e Alexa Fluor 647-conjugados de rato anti-ratinho Gr-1 anticorpos (0,05 ug / g BW em 100 ul de solução salina) através de uma veia jugular cannulus . </li><li> Vá para a barra de menu, e clique em &quot;Captura de Imagem&quot; na barra de ferramentas. </li><li> Um novo menu irá aparecer, em &quot;Imagem&quot;, selecione &quot;Fator de Bin&quot;, como 1x1 e selecione &quot;640&quot; na largura e &quot;460&quot; na altura. </li><li> Em &quot;Set Filter&quot;, marque a caixa de seleção para os canais que você gostaria de expor e definir o tempo de exposição para cada um. Por exemplo, o canal aberto: 10 ms, FITC canal: 50 ms, e Cy5 canal: 50 ms. </li><li> Selecione &quot;Capture Time-Lapse&quot; para ajustar o número de pontos de tempo e intervalo. For o modelo de inflamação, o número de pontos de tempo é definido como 1000-1500 com um intervalo de 200 msec (tempo decorrido Total: 5 min). </li><li> Clique em &quot;Test&quot; para ver uma imagem única avião no tempo de exposição especificado para aquele canal. Ajustar o tempo de exposição e / ou da luz do microscópio até que o histograma mostra uma distribuição de intensidade adequada (para multi-canal de captura, repetir esse processo para cada canal). </li><li> Depois de todos os parâmetros são definidos (canais, tempos de exposição, e assim por diante) no &quot;Captura de Imagem&quot;, selecione &quot;Iniciar&quot; para começar a captura. </li><li> Monitorar plaquetas rolantes e aderente e neutrófilos durante 5 min na metade superior de uma vénula cremaster inflamada no modelo de inflamação vascular. Oito a dez diferentes vénulas são gravados em um rato. </li><li> As imagens foram tiradas com uma ampliação total de 60X (1,0 NA objetivo imersão em água), dando um tamanho de janela de 157 x 118 mM mM. </li><li> Após a conclusão da experiência, os ratos foram sacrificados via cervical dislocation. </li></ol> 4. Análise de Dados para o Modelo de Inflamação venular <ol><li> Abra SlideBook software 5.0 e clique em &quot;Pasta&quot; na barra de menu para abrir um arquivo. </li><li> Clique no menu drop-down canal e selecione &quot;Abrir, FITC, Cy5, etc&quot; </li><li> Clique em &quot;renormalizar (barras vermelhas e verdes)&quot; e selecionar o canal. Arrastar as barras vermelhas e verdes para a esquerda ou para a direita a aproximadamente optimizar o sinal de fluorescência. </li><li> Clique em &quot;Aplicar&quot; e &quot;OK&quot; para atualizar a imagem. </li><li> Clique em &quot;Botão Miniatura&quot; para selecionar a exibição atual como padrão. </li><li> Jogar imagem timelapse capturado, e contar neutrófilos rolamento e aderentes durante o período de 5 min. </li><li> Para analisar a formação de trombos de plaquetas, vá para &quot;Máscara&quot; e selecione &quot;Criar&quot;. </li><li> Nome de uma máscara e marca &quot;Na imagem atual&quot;. </li><li> Clique em &quot;ícone do lápis Grande&quot; em &quot;Marquee Tool&quot; na tela principal. </li><li> Colorir uma região fora da embarcação na vista principal para definir uma máscara de fundo. </li><li> Ir to &quot;Máscara&quot;, clique em &quot;Copiar este plano&quot;, clique em &quot;Copiar máscara no ponto de tempo atual&quot; e selecione &quot;Todos os instantes&quot;, e clique em &quot;OK&quot;. </li><li> Para calcular sinal de fundo, vá em &quot;Estatísticas&quot; e clique em &quot;Estatísticas máscara&quot;. </li><li> Clique em &quot;Current Time Lapse 2D ou 4D Imagem (s)&quot; no Âmbito da imagem, e selecione &quot;máscara inteira&quot; no escopo de máscara. Em Recursos, selecione &quot;O tempo decorrido (hh: mm: ms)&quot; em data de captura, selecione &quot;Área (pixels)&quot; em Morfometria, selecione &quot;intensidade máxima&quot; em intensidade, e clique em &quot;Export&quot; (Este método estatístico que nos permite calcular automaticamente a intensidade de fluorescência (sinal de fundo)). </li><li> Abra o ficheiro de texto em MS Office Excel e calcular o valor médio do sinal de fundo durante todo o período de gravação. Esta é a intensidade de fluorescência de fundo. </li><li> Para determinar a intensidade do trombo, clique em &quot;ícone do lápis grande&quot; novamente no &quot;Marquee Tool&quot; na tela principal. </li><li> Cor dentro do vaso na vista principal para definir o sinal de plaquetas. </li><li> Vá em &quot;Máscara&quot;, clique em &quot;Copiar este plano&quot;, clique em &quot;Copiar máscara no ponto de tempo atual&quot;, selecione &quot;Todos os instantes&quot;, e clique em &quot;OK&quot;. </li><li> Vá em &quot;Estatísticas&quot; e clique em &quot;Estatísticas máscara&quot;. </li><li> Clique em &quot;Current Time Lapse 2D ou 4D Imagem (s)&quot; no Âmbito da imagem, e selecione &quot;máscara inteira&quot; no escopo de máscara. Em Recursos, selecione &quot;O tempo decorrido (hh: mm: ms)&quot; em data de captura, selecione &quot;Área (pixels)&quot; em Morfometria, selecione &quot;Intensidade Soma&quot; em intensidade, e clique em &quot;Export&quot; (Este método estatístico nos permitem o cálculo da intensidade de fluorescência no interior do vaso). </li><li> Abra o arquivo de texto no MS Office Excel. Sinal de fluorescência das plaquetas é calculado subtraindo-se o valor médio da área de fundo x intensidade (pixel) a partir da intensidade da soma do interior do vaso. Esta é a intensidade de fluorescência do trombo plaquetário. </li><li> Repetir este em 30 vénulas diferentes em 3-5 ratos diferentes. Em seguida, calcular o valor médio da intensidade de fluorescência das plaquetas. </li><li> Paraanalisar o rolamento dos neutrófilos e adesão, o influxo de rolamento dos neutrófilos foi determinada ao longo de 5 min em cada vénula e apresentados como o número de células por minuto. O número de neutrófilos aderentes que foram fixas durante&gt; 30 segundos e, lentamente, rastreado (&lt;10 fim ao longo de 30 s), mas não rolar, foram contados e apresentados como o número de células por 5 min. </li></ol> 5. Microscopia intravital para laser induzida trombose arteriolar 5-1 Calibração do laser de ablação <ol><li> Coloque uma corrediça espelhada em platina do microscópio, aplicar uma pequena gota de água destilada para a parte superior da lâmina, e ajuste da intensidade e da focagem utilizando a ocular. </li><li> Abrir &quot;janela de foco&quot; e selecione a opção &quot;FRAP&quot; guia na SlideBook software 5.0. </li><li> Definir a potência do laser em 40-50, e definir os dois &quot;repetições duplo clique&quot; e &quot;tamanho duplo clique&quot; para 8. Marque sobre o &quot;Guia&quot; caixa para abrir um conjunto de mira. </li><li> Selecione a opção &quot;Arrow&quot; ícone na barra de tarefas on parte superior da tela. </li><li> Sob o &quot;FRAP Alinhamento&quot;, clique em &quot;Fogo seguinte&quot;. Uma pequena mancha deve aparecer perto do canto inferior esquerdo do monitor. Uma vez que o local aparece, dê um duplo clique no centro dela. Uma vez clicado, outro ponto deve aparecer acima e à direita do primeiro ponto; clique duas vezes sobre ele. Repita este procedimento para os 16 pontos (o local 16 ª aparecerá no canto inferior direito). Clique em &quot;Salvar&quot; quando terminar de atualizar os parâmetros de calibração. </li><li> Para testar a precisão da calibragem, clique sobre o &quot;Centro&quot; local-um pequeno botão deve aparecer no centro da tela. Se não, repita a calibração até que a precisão desejada seja atingida. </li></ol> 5-2 laser induzida por formação de trombo arteriolar <ol><li> Colocar um rato anestesiado (ver o procedimento de 2-2 a 2-12) na platina do microscópio. </li><li> Sob a &quot;captura&quot; janela, selecione um protocolo ou criar um novo. As configurações são as seguintes: CY5 (70 exposição ms), Open (10 msec exposição) e FITC (50 exposição msec). Imagem é capturada mais de 20 min (aproximadamente 2.400 pontos de tempo com um intervalo de tempo de 500 ms). </li><li> Infundir Dylight 488-anti-rato conjugado e CD42c Alexa Fluor 647-conjugados anti-ratinho Gr-1 anticorpos, tal como descrito acima. </li><li> Otimizar os parâmetros de microscópio incluindo intensidade de fluorescência e imagem de campo claro como descrito em 3-3 para 3-7. </li><li> Clique no botão &quot;Teste&quot; para garantir a intensidade de campo claro adequada ou sinal de anticorpos, colocação arteríola e foco. </li><li> Clique em &quot;Iniciar&quot; para iniciar o processo de captura de imagem. </li><li> Antes de disparar o laser de ablação, certifique-se de que o ícone ponteiro do mouse foi selecionado e que a parede do reservatório está em foco claro. </li><li> Para disparar o laser, clique duas vezes em um ponto 2-3 mM internamente a partir da parede do vaso. Lesão das células endoteliais arteriolares provoca uma alteração de forma visual, que deve ser aparente na imagem de campo claro, seguindo-se a acumulação de plaquetas. Monitor de tromboformação, durante 5 min após a lesão laser. </li><li> Cinco minutos após a lesão de laser (do tamanho do trombo agora deve ser estável) pausar e cancelar a captura. Subsequentemente, iniciar uma captura com 2400 pontos de tempo com um intervalo de 500 msec para gravar plaquetas aderentes e neutrófilos rolantes / aderente durante 20 min. Três a cinco arteríolas são gravados em um rato. </li><li> Após a conclusão da experiência, os ratos foram sacrificados através de deslocação cervical. </li></ol> 6. Análise de Dados para o Modelo de Trombose arteriolar <ol><li> Vá passos 4,1 por 4,5. </li><li> Para obter a intensidade de fluorescência de fundo durante a formação de trombos de plaquetas, passar por cima de passos 4,7 por 4,14. </li><li> Vá em &quot;Máscara&quot;, clique em &quot;Segmento&quot;, selecione FITC no canal, e inserir o valor médio do sinal de fundo em baixa. Clique em &quot;Aplicar&quot; e &quot;OK&quot;. </li><li> Para a análise da intensidade de fluorescência de trombos plaquetários (Dylight 488-conjugado anticorpo anti-CD42c), </strong&gt; passar por cima de passos 4,18 por 4,19. Para o cálculo do sinal de anticorpo, &quot;Intensity Sum&quot; é subtraído do sinal de fundo (&quot;Intensidade máxima&quot; x &quot;da área (pixels)&quot;). Esta é a intensidade de fluorescência do trombo plaquetário. </li><li> Para quantificar os neutrófilos rolantes e aderente, desempenham o lapso de tempo e contar o número de células que visivelmente rolam ao longo dos trombos de plaquetas durante 20 min. Rolamento é definido como uma diminuição na velocidade dos neutrófilos ao interagir com o trombo de plaquetas durante pelo menos 2 segundos. Neutrófilos aderentes são definidos como quaisquer neutrófilos que permanecem ligados às trombo plaquetário durante pelo menos 2 min. O número de neutrófilos rolantes e aderente foi apresentada como o número de células por 20 min. Velocidade de rolamento foi calculado usando um sistema de rastreamento de partículas. </li></ol>

<ol>
	<li>Wagner, D. D., Frenette, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vessel+wall+and+its+interactions.">The vessel wall and its interactions.</a> Blood. 111, 5271-5281 (2008).</li><li>Nieswandt, B., Kleinschnitz, C., Stoll, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischaemic+stroke:+a+thrombo-inflammatory+disease.">Ischaemic stroke: a thrombo-inflammatory disease.</a> J. Physiol. 589, 4115-4123 (2011).</li><li>Yang, J., Furie, B. C., Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+P-selectin+glycoprotein+ligand-1:+its+role+as+a+selectin+counterreceptor+in+leukocyte-endothelial+and+leukocyte-platelet+interaction.">The biology of P-selectin glycoprotein ligand-1: its role as a selectin counterreceptor in leukocyte-endothelial and leukocyte-platelet interaction.</a> Thromb. Haemost. 81, 1-7 (1999).</li><li>Zarbock, A., Polanowska-Grabowska, R. K., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-neutrophil-interactions:+linking+hemostasis+and+inflammation.">Platelet-neutrophil-interactions: linking hemostasis and inflammation.</a> Blood Rev. 21, 99-111 (2007).</li><li>Chen, J., Lopez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+platelets+with+subendothelium+and+endothelium.">Interactions of platelets with subendothelium and endothelium.</a> Microcirculation. 12, 235-246 (2005).</li><li>Konstantopoulos, 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=Venous+levels+of+shear+support+neutrophil-platelet+adhesion+and+neutrophil+aggregation+in+blood+via+P-selectin+and+beta2-integrin.">Venous levels of shear support neutrophil-platelet adhesion and neutrophil aggregation in blood via P-selectin and beta2-integrin.</a> Circulation. 98, 873-882 (1998).</li><li>Maugeri, N., de Gaetano, G., Barbanti, M., Donati, M. B., Cerletti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+platelet-polymorphonuclear+leukocyte+interactions:+new+clues+to+the+antithrombotic+properties+of+parnaparin,+a+low+molecular+weight+heparin.">Prevention of platelet-polymorphonuclear leukocyte interactions: new clues to the antithrombotic properties of parnaparin, a low molecular weight heparin.</a> Haematologica. 90, 833-839 (2005).</li><li>Hidalgo, 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=Heterotypic+interactions+enabled+by+polarized+neutrophil+microdomains+mediate+thromboinflammatory+injury.">Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury.</a> Nat. Med. 15, 384-391 (2009).</li><li>Cho, J., Furie, B. C., Coughlin, S. R., Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+extracellular+protein+disulfide+isomerase+during+thrombus+formation+in+mice.">A critical role for extracellular protein disulfide isomerase during thrombus formation in mice.</a> J. Clin. Invest. 118, 1123-1131 (2008).</li><li>Cho, J., 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=Protein+disulfide+isomerase+capture+during+thrombus+formation+in+vivo+depends+on+the+presence+of+beta3+integrins.">Protein disulfide isomerase capture during thrombus formation in vivo depends on the presence of beta3 integrins.</a> Blood. 120, 647-655 (2012).</li><li>Gross, P. L., Furie, B. C., Merrill-Skoloff, G., Chou, J., Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-versus+microparticle-mediated+tissue+factor+transfer+during+arteriolar+thrombus+development.">Leukocyte-versus microparticle-mediated tissue factor transfer during arteriolar thrombus development.</a> Journal of Leukocyte Biology. 78, 1318-1326 (2005).</li><li>Falati, S., Gross, P., Merrill-Skoloff, G., Furie, B. C., Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+in+vivo+imaging+of+platelets,+tissue+factor+and+fibrin+during+arterial+thrombus+formation+in+the+mouse.">Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse.</a> Nat. Med. 8, 1175-1181 (2002).</li><li>Barthel, S. 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=Alpha+1,3+fucosyltransferases+are+master+regulators+of+prostate+cancer+cell+trafficking.">Alpha 1,3 fucosyltransferases are master regulators of prostate cancer cell trafficking.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 19491-19496 (2009).</li><li>Trzpis, M., McLaughlin, P. M., de Leij, L. M., Harmsen, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+adhesion+molecule:+more+than+a+carcinoma+marker+and+adhesion+molecule.">Epithelial cell adhesion molecule: more than a carcinoma marker and adhesion molecule.</a> The American Journal of Pathology. 171, 386-395 (2007).</li><li>Junt, T., 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=Dynamic+visualization+of+thrombopoiesis+within+bone+marrow.">Dynamic visualization of thrombopoiesis within bone marrow.</a> Science. 317, 1767-1770 (2007).</li><li>Egan, C. E., Sukhumavasi, W., Bierly, A. L., Denkers, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+multiple+functions+of+Gr-1(+)+cell+subpopulations+during+microbial+infection.">Understanding the multiple functions of Gr-1(+) cell subpopulations during microbial infection.</a> Immunologic Research. 40, 35-48 (2008).</li></ol>

Os autores declaram que não há interesse competindo financeira.

Aqui nós descrevemos um protocolo detalhado em tempo real a microscopia de fluorescência intravital para visualizar heterotípicos plaquetas neutrófilos interações sobre o endotélio ativado durante a inflamação vascular e trombose. Anteriormente, semelhantes de fluorescência abordagens microscópicas foram relatados para estudar o mecanismo molecular da formação de trombos e de inflamação vascular. 8,12 Uma vez que a interacção célula-célula heterotípica pode ser importante para a vaso-oclus...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>7447-40-7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>10035-04-8</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2 6H2O</td>
			<td>Fisher Scientific</td>
			<td>7791-18-6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher Scientific</td>
			<td>144-55-8</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.9% NaCl Saline</td>
			<td>Hospira</td>
			<td>0409-4888-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Hospira</td>
			<td>0409-2051-05</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Lloyd</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intramedic Tubing (PE 90)</td>
			<td>BD Diagnostics</td>
			<td>427421</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intramedic Tubing (PE 10)</td>
			<td>BD Diagnostics</td>
			<td>427401</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Murine TNF-&alpha;</td>
			<td>R&amp;D Systems</td>
			<td>410-MT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dylight 488- labeled rat anti-mouse CD42b antibody</td>
			<td>Emfret Analytics</td>
			<td>X488</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647-conjugated anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody</td>
			<td>BioLegend</td>
			<td>108418</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NESLAB EX water bath/circulator</td>
			<td>Thermo-Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olympus BX61W microscope</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TH4-100 Power</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lambda DG-4</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MPC-200 multi-manipulator</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ROE-200 stage controller</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>C9300 high-speed camera</td>
			<td>Hamamatsu</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intensifier</td>
			<td>Video Scope International</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ablation Laser</td>
			<td>Photonic Instruments, Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SlideBook 5.0</td>
			<td>Intelligent Imaging Innovations</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

real-time imaging of heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice

Interacção de plaquetas e leucócitos activados (sobretudo neutrófilos) no endotélio activado trombose e medeia a inflamação vascular. 1,2 Durante a formação do trombo no sítio da lesão arteriolar, plaquetas aderentes ao endotélio activado e proteínas da matriz subendotelial suporte de rolamento dos neutrófilos e adesão 3. Por outro lado, sob condições inflamatórias venulares, os neutrófilos aderentes ao endotélio activado pode suportar a adesão e acumulação de plaquetas circulantes. Agregação de plaquetas de neutrófilos heterotípica requer processos sequenciais pelas interacções específicas do receptor de contra-receptor entre as células. 4 Sabe-se que células endoteliais activadas libertar as moléculas de adesão, tais como o factor de von Willebrand, iniciando assim a adesão de plaquetas e acumulação sob condições de alto cisalhamento. 5 Além disso, células endoteliais ativadas apoiar rolamento e adesão de neutrófilos por selectinas expressando umad molécula-1 de adesão intercelular (ICAM-1), respectivamente, sob condições de baixo cisalhamento. Platelet 4 P-selectina interage com os neutrófilos a P-selectina glicoproteína ligando-1 (PSGL-1), induzindo deste modo a activação de neutrófilos β2 integrinas e firmes a adesão entre dois tipos de células. Apesar dos avanços nas experiências in vitro em que heterotípicas plaquetas de neutrófilos interacções são determinados em sangue completo ou células isoladas, 6,7 desses estudos não é possível manipular as condições de stress oxidante durante a doença vascular. Neste relatório, utilizando fluorescentemente marcados, os anticorpos específicos contra um rato de plaquetas e neutrófilos marcador, nós descrevemos um protocolo detalhado microscópica intravital para monitorar interacções heterotípicas de plaquetas e neutrófilos no endotélio activado durante TNF-α induzida por inflamação ou seguindo induzida por laser lesão no músculo cremaster microvasos de camundongos vivos.

Usando a análise de microscopia intravital detalhada, heterotípicas plaquetas de neutrófilos interações no endotélio activado foram visualizadas por infusão de anticorpos marcados com fluorescência contra uma plaqueta (CD42c) ou marcador de neutrófilos (Gr-1) em murganhos vivos. Em um modelo de TNF-α induzida por inflamação venular, neutrófilos rolantes foram estavelmente mais aderido ao endotélio, presumivelmente através da interacção de β2 integrinas activadas com ICAM-1 ...

Watch this Scientific Journal Video about Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mic

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Aqui descrevemos uma técnica experimental de microscopia de fluorescência intravital para visualizar heterotípicos plaquetas neutrófilos interações sobre o endotélio ativado durante a inflamação vascular e formação de trombos em camundongos vivos. Esta tecnologia microscópico será valiosa para estudar o mecanismo molecular da doença vascular e para testar agentes farmacológicos, em condições fisiopatológicas.

Imagens em tempo real de heterotípica plaquetas neutrófilos Interações sobre o endotélio ativado durante a inflamação vascular e formação de trombos em camundongos vivos

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

real-time-imaging-heterotypic-platelet-neutrophil-interactions-on

Research

JoVE Journal

Immunology and Infection

15.6K visualizações.  University of Illinois at Chicago. Aqui descrevemos uma técnica experimental de microscopia de fluorescência intravital para visualizar heterotípicos plaquetas neutrófilos interações sobre o endotélio ativado durante a inflamação vascular e formação de trombos em camundongos vivos. Esta tecnologia microscópico será valiosa para estudar o mecanismo molecular da doença vascular e para testar agentes farmacológicos, em condições fisiopatológicas.

Vídeo: Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

Em tempo real de imagens de leucotrienos B 4 Migração mediada por células e BLT1 Interações com β-arrestina

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

Imunologia e Infecção

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

Imagem adesão leucocitária ao endotélio vascular na pressão intraluminal de alta

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Detecção de MicroRNAs em Microglia por PCR em tempo real no SNC normal e durante Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Em tempo real de imagens ao vivo de células T Sinalização de Formação de Complexos

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

Imagens em tempo real de endoteliais junções célula-célula durante Neutrophil Transmigração Sob Fisiológica Fluxo

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

A Novel<em&gt; In vitro</em&gt; Modelo para estudar as interações entre a Whole Humano Sangue e endotélio

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Decifrar e Imagem Patogênese e Cording de<em&gt; Mycobacterium abscessus</em&gt; Em embriões Zebrafish

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

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 ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

Caracterização metabólica de Polarizada M1 e M2 macrófagos da medula óssea derivadas usando análises em tempo real do fluxo extracelular

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

Modelo de doença microfluida in vitro para estudar interações endoteliais de sangue inteiro e dinâmica do coágulo sanguíneo em tempo real

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...