This work was supported by EMBO (to Y.E.), Foundation Machaon (to Y.E.) and SNSF (to B.A.I.). We thank the Bioimaging Core Facility for the availability of the Nikon A1r microscope and technical assistance. We thank Mrs. Clarissa Bartley for English correction.

NOTA: os procedimentos com animais foram realizados de acordo com o Comitê de Ética Institucional Animal Care, em Genebra, Suíça e Veterinário Cantonal. Número de autorização GE / 63/14. 1. Preparação de uma suspensão de células isoladas a partir da medula <ol><li> Sacrifício rato (8-12 semanas de idade) por deslocamento cervical. Esterilizar patas traseiras com etanol 70%. Retire a pele das pernas traseiras. </li><li> Dissecar fêmures e tíbias do mouse e remover o tecido de pernas com um bisturi. Lavar os pés com etanol 90% e coloque em um prato de cultura cheio com PBS. </li><li> Sob o capô cultura, tecido limpo de o fémur ea tíbia, com um bisturi e separada na articulação do joelho. Cortar cada extremidade dos ossos. </li><li> Lavar medula óssea a partir de cada osso, utilizando uma agulha de 23G e uma seringa de 1 ml cheio com meio de preparação (sem vermelho de fenol DMEM / F12, 1% de soro de rato), para um tubo de 50 ml de parafuso. Disrupt agregados celulares por delicadamente passaging através do ne 23Gedle e uma seringa de 1 ml. Levar o volume total a 25 ml com meio de preparação. </li><li> Centrifugar a 250 xg, 5 min, 4 ° C. Sobrenadante de descarte. Pelete ressuspender em 1 ml de glóbulos vermelhos de tampão de lise RBLC (8,3 g de NH 4 Cl, 1 g de NaHCO 3, 1 ml de EDTA (100 mM), completar para 1 L com água destilada, filtro de 0,22 um) em uma de 15 ml de parafuso tubo. Incubar 30 segundos em gelo. </li><li> Adicionar 10 ml de meio e centrifuga-se a preparação de 250 x g, 5 min, 4 ° C. Descartar o sobrenadante e ressuspender em 2 ml de meio de preparação. </li></ol> 2. Neutrófilos Enriquecimento por selecção negativa <ol><li> Adicionar 150 mL de Rato neutrófilos enriquecimento cocktail (kit plataforma de isolamento de células, tais como EasySep). Misturar e incubar em gelo durante 10 min. </li><li> Adicionar 10 ml de fenol vermelho DMEM livre. Inverter e centrifugar uma vez a 250 x g, 3 minutos, 4 ° C. </li><li> Sobrenadante de descarte. Pellet ressuspender em 1,75 ml de meio de preparação em 5 ml de poliestireno rodada tubos de fundo. Adicionar 250 ul BiotCocktail na Seleção. Misturar e incubar em gelo durante 10 min. </li><li> Vortex as partículas magnéticas (do kit de isolamento de células de plataforma) durante 30 segundos para ressuspender as partículas. Adicionar 500 ul de partículas magnéticas a suspensão de células. Misturar e incubar em gelo durante 10 min. </li><li> Colocar o tubo em o íman. Espere 3 min. </li><li> Inverter o ímã em um novo 5 ml de poliestireno rodada tubos de fundo para recolher as células não marcadas desejados. Não tome a última gota pendurada no tubo. As células indesejadas permanecerá no tubo dentro do magneto. Descarte este tubo em um resíduos de risco biológico. </li><li> Coloque o novo tubo no ímã. Espere 3 min. </li><li> Inverter o ímã para um novo 15 ml parafuso tubo para recolher as células não marcadas desejados. Não tome a última gota pendurada no tubo. </li><li> Volume completo até 5 ml com DMEM sem fenol vermelho. </li></ol> 3. Marcação de Neutrófilos <ol><li> Adicionar 0,5 mL de um corante laranja, tais como celular Rastreador Orange (concentração de trabalho1 ï¿½, CMRA - clorometil- rodol-acetato, estoque 10 mM em DMSO). Incubar 2 min a 37 ° C. </li><li> Adicionar 2,5 ml de meio de preparação. Centrifugar a 250 xg, 5 min, 4 ° C. </li><li> Sobrenadante de descarte. Pelete Ressuspender em 1 ml de meio de preparação em um tubo de 1,5 ml. </li><li> Tomar uma alíquota de contar com hematocytometer. </li><li> Incubar 5 min a 37 ° C. Centrifugar a 250 xg, 5 min, 4 ° C. </li><li> Sobrenadante de descarte. Ressuspender o sedimento em 1 ml de PBS. Centrifugar a 250 xg, 5 min, 4 ° C para lavar. </li><li> Sobrenadante de descarte. Ressuspender o granulado como 10x10 6 células por 100 ul de PBS. Manter em gelo até injeção iv. NOTA: as células têm de ser injectados iv tão rapidamente quanto possível. 6-12x10 6 neutrófilos / rato são normalmente obtidos. </li></ol> 4. Rato Preparação para Microscopia intravital NOTA:.. Etapa 4.1) e 4.2) deve ser realizada no início da experiência para evitar tempo de espera prolongado de neutrófilos marcados na ICe. <ol><li> Pesar o CX 3 CR1 rato gfp (8-12 semanas de idade). </li><li> Preparar 800 ul de anestésicos (cetamina 60 mg / kg, xilazina 4,5 mg / kg, acepromazone 1,75 mg / kg em PBS). </li><li> Anestesiar mouse. Ip injeção com 200 mL / 20 g de rato. Anestesia controle por toe beliscar e verificando a freqüência respiratória. </li><li> Injectar neutrófilos (10x10 6 células em 100 ul de PBS) por via intravenosa na veia da cauda usando laterais ou do seio retro-orbital, para a conveniência do experimentador. </li><li> Coloque o mouse sobre almofada de aquecimento e proteger os olhos com gel ocular. </li><li> Uma lamela de vidro (35 mm de diâmetro) é colocado em cerca de 10 cm prato de cultura de tecido, que tem um orifício de diâmetro de 30 mm. O uso de óleo é o de manter a placa de cultura de tecido em contacto com a lamela. </li><li> Inciso pele abdominal com uma tesoura para expor o peritônio. Inciso peritônio com uma tesoura para expor intestino. </li><li> Adicionar 200 ul de PBS (pré-aquecida a 37 ° C) para a pcavidade eritoneal. Leve o mouse na mão e transformá-lo virado para baixo, de modo que o intestino fica fora da cavidade peritoneal com uma pressão suave. Posicione o mouse na placa de cultura de tecidos. </li><li> Gentilmente deroll intestino com cotonetes esterilizados para a lamela, a fim de expor os vasos mesentéricos. </li><li> Corte lenços de papel em bandas e os molhe com 37 ° C aquecida PBS. Imobilizar o intestino com os pedaços de tecidos de papel para reduzir os movimentos resultantes do peristaltismo. </li><li> Coloque o prato de cultura de tecidos e o rato dentro da câmara termostatizada 37 ° C para o palco bandeja feito por encomenda do microscópio invertido. Introduzir uma seringa contendo anestésicos sc na pata traseira. NOTA: Para além disso, o rato pode receber oxigénio (0,5 L / min) com uma máscara. </li><li> Controle do mouse para anestesia por beliscar dedo do pé e verificar a freqüência respiratória a cada 30 min. Se necessário, fornecer um impulso de anestésicos (20 l) para manter adequadamente anestesia. NÃOE: A secagem da vasculatura deve ser evitada. Por isso, a sua humidade é mantida por molhagem regularmente os lenços de papel usados ​​para imobilizar o intestino com 37 ° C aqueceu-PBS. </li></ol> 5. Medição de neutrófilos e monócitos Comportamento no Vessels inflamadas in vivo utilizando Microscopia intravital <ol><li> Definir lasers para 488- 561- e com o menor poder necessário para evitar fototoxicidade induzida por laser. Nas nossas experiências, é inferior a 0,5%. Intensidade da GFP em monócitos e corante laranja em neutrófilos é tão brilhante que geralmente 0,3% é suficiente. NOTA: Isto representa uma potência de laser de 0,15-0,25 mW com o nosso sistema. </li><li> Adquirir imagens de 512 x 512 pixels (ou seja, 635 um) em 2,2 segundos com uma pinhole aberto usando o 20X objetivo seco do microscópio invertido. Usar uma profundidade Z entre 10 e 20 um, que garante um sinal suficientemente com as baixas potências de laser. NOTA: Nós geralmente realizar experimentos com um z-profundidade entre 12 a 1656; m. No caso de filmes e figuras apresentadas, a profundidade Z é de 20 uM. O contraste de fase permite a identificação de paredes endoteliais e formação de imagens é executada no mesentério. Dado que as células de patrulha em se movem lentamente com uma velocidade de 5-10 uM / min 6, a gravação de um campo de interesse cada 20 segundos é satisfatória. Com as configurações descritas, o estágio motorizado permite a gravação de até 7 campos de interesse, ao mesmo tempo. </li><li> Colocar o rato e os vasos mesentéricos termostato na câmara do microscópio são lhes permitir recuperar a partir da preparação durante 30 minutos antes da aquisição do primeiro filme. Durante este tempo, escolher vários domínios de interesse. Concentre-se na superfície luminal do vaso para o objectivo mais próximo. Se necessário, aproveitar o ligeiro auto-fluorescência do endotélio para ajustar o foco. NOTA: A cirurgia durante a preparação do mouse ligeiramente salienta o endotélio. Consequentemente, rolando de neutrófilos e/ ou monócitos pode ser observado na primeira a seguir à preparação de 15 min rato. </li><li> Grave um primeiro filme durante 30 minutos sob condições de estado estacionário, uma imagem a cada 20 segundos. NOTA: O software adquire cada campo de interesse em 2.2 segundos e o estágio motorizado move-se automaticamente de um campo para o outro. Ele vai voltar para a primeira posição no prazo de 20 segundos, durante os 30 minutos do filme gravação. </li><li> Para iniciar a inflamação dos vasos sanguíneos, adicionar uma gota de 20 ul de PBS contendo 100 ug de TLR2 / TLR1 agonista Pam3CSK4 12 directamente sobre os vasos trabalhada. </li><li> Controlar o foco para cada área de interesse. NOTA: Durante o curso de aquisição, há sempre pequenas alterações de concentração no eixo z. Apesar do 10-20 jiM z profundidade, isto pode resultar num decréscimo da intensidade de fluorescência. Portanto, reorientar manualmente cada campo de juros no final de cada filme, se necessário. Grave filmes durante 30 min-períodos e até 3 horas paraTal após o início da inflamação. </li><li> No final da experiência, o sacrifício do rato anestesiado por deslocamento cervical. </li></ol> 6. Geração de arquivos de filme e Rastreamento de leucócitos NOTA: O software de aquisição Nikon gera .nd2 arquivos, mas o procedimento a seguir seria semelhante com outros softwares e marcas. <ol><li> Ficheiros de exportação como a série de tiff. </li><li> Ir para software ImageJ <a href="http://rsb.info.nih.gov/ij/">(http://rsb.info.nih.gov/ij/).</a> Importar a série de tiff. Cada canal como um arquivo diferente. </li><li> Aplicar um filtro mediano com um raio de 2,0 pixel para os canais verde e vermelho para reduzir o ruído de fundo (Processo&gt; Filtros&gt; medianos ...). </li><li> Converta arquivos em binário (Processo&gt; binário&gt; Faça binário). Para ativar o software para calcular o limite para cada imagem. </li><li> Mesclar canais em uma série de imagens (Imagem&gt; Cor&gt; Mesclar Canais ...). Selecione os monócitos no green canal, os neutrófilos no canal vermelho e da luz transmitida no canal cinza. </li><li> Converta imagens empilhados em cores RGB (Imagem&gt; Cor&gt; pilha para RGB). </li><li> Salve o arquivo como .avi </li><li> Os dados são importados e compilado no software Imaris (Bitplane). Movimentos de monócitos e neutrófilos são, então, rastreados usando o Assistente de Rastreamento Spot. </li></ol>

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The authors declare that they have no competing financial interests.

As metodologias descritas neste manuscrito fornecer uma abordagem consistente para estudar de forma eficiente monócitos e comportamento de neutrófilos nas veias mesentérica, em condições de estado estacionário e inflamatórias. O passo crucial da preparação é a imobilização de tecidos do intestino com PBS-humedecidos. Se realizada corretamente, vasos mesentéricos são bem expostos na lamela para aquisição de imagem. Isso permite a seleção de vários domínios de interesse par...

<html> Os neutrófilos e os monócitos são células do sistema imune inato que continuamente circulam no sangue. Após a lesão ou infecção, os sinais inflamatórios induzir diapedese de leucócitos em tecidos danificados e infectadas, aqui iniciar uma resposta imune celular 1-3. A rapidez da mobilização de leucócitos determina o resultado positivo das respostas imunes. Estes processos intrincados contar com moléculas específicas (por exemplo, selectinas, quimiocinas vinculado do endotélio) presentes no endotélio inflamado que ajudam para o estabelecimento de contactos adesivas entre leucócitos circulantes e do endotélio 1-3 .Para obter insights sobre as moléculas implicadas na leucócitos recrutamento cascata, é importante para visualizar a cinética do recrutamento de células e para controlar o comportamento de cada célula / população. Um método eficaz envolve microscopia intravital em ratinhos transgénicos que expressam proteínas fluorescentes em células de interesse. conteúdo &quot;&gt; Até à data, várias abordagens utilizando microscopia intravital foram desenvolvidos para a imagem da vasculatura 4,5. Por exemplo, a imagem latente da derme ou veias da orelha vasculatura mesentéricos por microscopia confocal intravital foi usada para identificar o comportamento de patrulha Ly6C murino e humano monócitos baixos CD14 dim CD16 + monócitos no lado luminal dos vasos sanguíneos sob condições de estado estacionário 6-8. O modelo de vasculatura do mouse cremaster é muitas vezes usado para monitorar o comportamento dos neutrófilos ou monócitos Ly6C inflamatória elevadas em condições inflamatórias ou isquêmicas em ratinhos transgénicos. Nesse caso, cremaster é estimulada através de injecção intrascrotal de IL1β, CCL2, TNFp ou fMLP. Depois de 4/2 hora, os tecidos são então exteriorizado e analisados ​​por microscopia confocal intravital 9-11 cirurgicamente. O protocolo seguinte descreve um método para monitorizar monócitos e neutrófilos, ao mesmo tempo com qualquerinvertido de fluorescência confocal. Além disso, o nosso método fornece detalhes sobre como imagem do mesmo navio antes (condição de estado estacionário) e, após a inflamação e como seguir a cinética do recrutamento de leucócitos. Para este fim, usamos o CX 3 CR1 rato gfp / peso, cujo monócitos expressam eGFP, iv injetado com uma laranja neutrófilos murino marcadas com corante. Utilizando um microscópio invertido confocal, é possível (1) para monitorizar e analisar os monócitos patrulha Ly6C baixas sob condições de estado estacionário e (2) para seguir o recrutamento de monócitos e neutrófilos no mesmo vaso, após a inflamação local. Aqui nós criamos as condições inflamatórias usando o agonista TLR2 / TLR1 Pam3CSK4 12. Além disso, tal imagem pode ajudar a determinar o papel de moléculas específicas de interesse nos vários passos da cascata de recrutamento de leucócitos se realizada em ratinhos knock-out específicas ou na presença de anticorpos bloqueadores 6,9,13.

<table>
	<tbody>
		<tr>
			<td>5 mL polystyrene round bottom tubes&#160;</td>
			<td>Beckton Dickinson</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>10x CFI Plan Apochromat 0.45&#160;&#160; DT:4mm&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20x CFI Plan Apochromat VC 0.75&#160;&#160; DT:1mm&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Tracker Orange CMRA Dye</td>
			<td>Life Technologies</td>
			<td>C34551</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>Stem Cell Technologies</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Mouse Neutrophil Enrichement kit</td>
			<td>Stem Cell Technologies</td>
			<td>19762</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>PAA</td>
			<td>A15-042</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion Oil Type A</td>
			<td>Nikon</td>
			<td></td>
			<td>any viscous oil&#160;</td>
		</tr>
		<tr>
			<td>Life Box Temperature Control System</td>
			<td>Life Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sigma Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R confocal microscope</td>
			<td>Nikon</td>
			<td>A1R</td>
			<td>inverted microscope, motorized x/y/z stage, NIS elements software</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>phenol red free DMEM/F12</td>
			<td>Life Technologies</td>
			<td>21041-025</td>
			<td>any phenol red free medium is suitable</td>
		</tr>
		<tr>
			<td>PAM3CSK4</td>
			<td>Invivogen</td>
			<td>tlrl-pms</td>
			<td>reconstitute in PBS</td>
		</tr>
		<tr>
			<td>Rat serum</td>
			<td>Stem Cell Technologies</td>
			<td></td>
			<td>included in EasySep Mouse Neutrophil Enrichement kit</td>
		</tr>
		<tr>
			<td>tissue culture dish 100</td>
			<td>TPP</td>
			<td>93100</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging neutrophils and monocytes in mesenteric veins by intravital microscopy on anaesthetized mice in real time

Resposta imunitária eficaz é dependente rápida mobilização de leucócitos do sangue para o local da infecção ou lesão. Investigar a migração de leucócitos in vivo é essencial para compreender a base molecular da migração transendotelial de leucócitos e a interacção com o endotélio vascular. Uma poderosa abordagem envolve microscopia intravital em ratinhos transgénicos que expressam proteínas fluorescentes em células de interesse. Aqui apresentamos um protocolo para monócitos de imagem e neutrófilos no CX 3 CR1 gfp / p iv rato injetado com neutrófilos marcados com corante laranja com um microscópio confocal invertido. Filmes de lapso de tempo recolhidas a partir de 30 minutos a várias horas de imagens permitem a análise do comportamento de leucócitos nas veias mesentérica em ambas as condições estaduais e inflamatórias estáveis. Nós também descrevem os passos para induzir localmente inflamação dos vasos sanguíneos com TLR2 / TLR1 agonista Pam3SK4 e monitorar o subsequeNT recrutamento de neutrófilos e monócitos. A técnica apresentada pode também ser utilizado para monitorizar outras populações de leucócitos e as moléculas implicadas na investigar o recrutamento de leucócitos ou o tráfico que utilizam outros estímulos ou ratinhos transgénicos.

O manuscrito descreve um protocolo optimizado para controlar facilmente o comportamento dos monócitos e neutrófilos no mesentério de ratos anestesiados em tempo real. A utilização de uma câmara de 37 ° C-termostatizada é obrigatório para manter a temperatura do rato e também devido à temperatura de movimento dependentes dos leucócitos. Preparação do rato é apresentada na Figura 1. A Figura 2 mostra toda a área visto ao microscópio. Luz transmitida permite a identificaç...

Watch this Scientific Journal Video about Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time at JoVE.com

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

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 ...

imaging-neutrophils-monocytes-mesenteric-veins-intravital-microscopy

Research

JoVE Journal

Immunology and Infection

10.4K visualizações.  University of Geneva. 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.

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

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

Rastreamento Crawling neutrófilos Intraluminal, migração transendotelial e Quimiotaxia em tecido por Microscopia intravital Vídeo

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Imunologia e Infecção

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

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

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 ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

Estudo das interacções de<em&gt; Staphylococcus aureus</em&gt; Com neutrófilos por citometria de fluxo e Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

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 ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Imagens em tempo real de células mielóides em Dynamics<em&gt; Apc<sup&gt; Min / +</sup</em&gt; Intestinal Tumores girando Disk microscopia confocal

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using 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, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

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, ...

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 ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Imagens ao vivo de antifúngicos Atividade por neutrófilos humanos primários e monócitos em resposta à<em&gt; A. fumigatus</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

Interação de célula de imagem latente na Mucosa traqueal durante a infecção de vírus de Influenza usando microscopia Intravital dois fotões

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease.
We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section.
IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

Infiltração de leucócitos do Músculo Cremaster em Camundongos Avaliados por Microscopia Intravital

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...