Este trabalho foi financiado por uma bolsa de investigação da Canadian Institutes of Health Research (CIHR, MOP-86749). L. Liu é um receptor de CIHR New Investigator Award (MSH-95374).

1. Preparação de quimioatraente em gel de agarose <ol><li> Pipeta de 10 mL de 2 × PBS em um tubo cônico de 50 ml, e aquecer o tubo colocando-o em um copo contendo água quente. </li><li> Pipetar 10 mL de água destilada e adicionar 0,4 g de pó de agarose em outro tubo cônico de 50 mL (com a tampa ligeiramente soltos) e aquecer a mistura ferver até ficar em um forno de microondas (por 1 min ~ em um forno de microondas 700-watt) . </li><li> Adicione o aquecido 2 × PBS para a solução de agarose tubo, redemoinho solução mista e mantê-lo aquecido no copo de água quente. </li><li> Micropipeta solução chemoattractant (por exemplo, 10 ml de 0,5 mg de CXC quimiocinas MIP-2 ou 12 mL de uma WKYMVm mM) na tampa de um tubo Eppendorf de 1,5 mL contendo 3 mL de tinta da Índia e misture bem por aspiração utilizando uma micropipeta (evitar bolha de ar). </li><li> Corte a extremidade da ponta de uma pipeta de 200 mL e 110 mL micropipeta de agarose solução (42 ° C) na tampa e imediatamente misture bem utilizando outra ponta da pipeta (evitar bolhas de ar). </li><li> Armazenar o gel chemoattractant contendo a +4 ° C. </li></ol> 2. Preparação de músculo Cremaster para Microscopia intravital (Figura 1) <ol><li> Anestesiar um rato macho adulto por uma injeção ip de uma mistura de xilazina 10 mg / kg e 200 mg de cloridrato / kg cetamina. </li><li> Raspar a área ao longo da veia jugular externa direita e face anterior do escroto com um barbeador elétrico. Após a anestesia, é importante dar atenção e cuidados especiais para o rato anestesiado. A lâmpada de calor pode ser usado para impedir o mouse de hipotermia. O mouse deve estar livre de dor reflexa. </li><li> Faça uma incisão horizontal, encontrar e cateterização da veia jugular utilizando um tubo PE-10 preenchido com solução salina com heparina 100 U / mL. Cateterismo da veia jugular é necessário para a administração de anestésicos e medicamentos adicionais, quando necessário. </li><li> Fixar as pernas traseiras do mouse com fita umbilical com o mouse deitado de barriga para cima em um home-made bordo do músculo cremaster (Figura 1). </li><li> Conectar a placa para um circulador de água 37 ° C para manter o músculo cremaster eo corpo do mouse quente. </li></ol> Nota: Todos os procedimentos 2,6-2,14 deve ser realizada muito gentilmente 5. <ol start="6"><li> Faça uma incisão na pele escrotal para expor o músculo cremaster esquerda. Dissecar cuidadosamente o músculo da fáscia associados. </li><li> Superfuse o músculo cremaster com 37 ° C-aquecido bicarbonato-buffered saline (131,9 NaCl, 4,7 KCl, 1,2 MgSO 4, 20 NaHCO 3, em mM, pH 7,4) usando uma bomba peristáltica. </li><li> Amarre uma sutura 4-0 para a extremidade distal do músculo cremaster para segurá-la para baixo sobre o pedestal de vidro clara visualização da placa do músculo cremaster. </li><li> Cauterizar o músculo cremaster longitudinalmente. Com sutura 4-0, mantenha o plano muscular e fixá-lo ao longo das bordas sobre o pedestal. Separar o testículo e epidídimo do músculo subjacente e movê-los para dentro da cavidade abdominal. </li><li> Superfuse o músculo em ~ 0,6 ml / min com o tampão de perfusão 37 ° C-aquecido e cobrir o músculo exposto com uma lamela de vidro 22 × 22 mm. </li><li> Coloque a placa do músculo cremaster no palco microscópio, examine o músculo sob o microscópio, encontrar uma vênula adequado postcapillary (Selecione a vênula que é reto e não ramificados e tem taxa de cisalhamento normal e um diâmetro de 25-40 mm) e ajustar a câmera de vídeo para permitir a vênula para ser visualizado em uma posição vertical em ambos os lados esquerdo ou direito do monitor de TV. </li><li> Após 30 min de equilíbrio, a gravar as imagens de vídeo da vênula selecionados postcapillary por 5 min como dados de base de controle utilizando um gravador de vídeo. </li><li> Parar o superfusão e retire a lamínula sobre o músculo. </li><li> Coloque um ~ 1-3 mm de tamanho gel chemoattractant contendo na superfície do músculo cremaster em uma área pré-selecionadas 350 mM de e paralela à vênula observado postcapillary, adicione lamela para segurar o gel no local e superfuse o tecido muscular em muito ritmo lento (≤ 10 mL / min) para permitir o estabelecimento de um gradiente de chemoattractant que é liberada lentamente e formado a partir do gel. </li><li> Gravar a imagem de vídeo de 90 min após a adição do gel contendo chemoattractant. Durante a gravação, ajustar e manter o foco microscópio sobre a aderir, rastejando, transmigrando e chemotaxing leucócitos dentro da vênula e no tecido muscular. </li><li> Após a experiência, importe o arquivo de vídeo para um computador para análise. </li></ol> 3. Rastreamento de células Usando ImageJ <ol><li> Em um computador, extrair e converter o vídeo para o formato AVI (por exemplo, use software livre bitRipper computador para converter vídeo de DVD para arquivos AVI). </li><li> Use software de edição de vídeo para gerar o filme tempo decorrido. Por exemplo, use o Windows Movie Maker para fazer um filme tempo decorrido (até 1 / 512 ou 1 / 1024 tempo-lapsot taxa de 30 fps) a partir do original, vídeo em tempo real. Converter e salvar o filme tempo decorrido sem compressão de DV-AVI formato. </li><li> Gravar as imagens de um micrômetro de calibração sob o microscópio mesma configuração, importar imagens para o computador, abra as imagens com ImageJ. No ImageJ, o total de pixels aparecem no canto superior esquerdo da tela (por exemplo, 720 × 480 pixels). Medir o tamanho da tela em ambos os eixos X e Y (por exemplo, 200 × 150 mm). A partir daí, calcular o número de pixels por M (por exemplo, x = 720 / 200 = 3,6 pixels / M, e y = 480 / 150 = 3,2 pixels / mm). </li><li> Para importar o filme, abra ImageJ novamente, clique em &quot;File-Import-Usando Movies Quicktime Plug-in&quot;, selecionar o filme a ser analisado e clique em &quot;OK&quot; na interface de &quot;abridor de Filme QT&quot;. </li><li> Clique em &quot;Rastreamento Plugins-Manual&quot; para rastrear células. Preencha as informações relevantes para os campos no fundo antes de iniciar o rastreamento. Brevemente, <ol><li> Intervalo de tempo (em seg) = fold-time-lapse/30 (Por exemplo, um 1020 × lapso de tempo seria 1020-1030 = 34 intervalo de seg / frame). </li><li> x / y = calibração da medição M / pixel usando a calibração da imagem do micrômetro. </li><li> z = 0 calibração (como o mouse músculo cremaster é uma camada extremamente fina de tecidos e células rastejando e migração são aproximadamente 2D sob brilhante-campo transiluminação). </li><li> Pesquisa tamanho quadrado para centrar = 1. </li><li> Dot size / Linha de largura / Tamanho da fonte: eles podem ser ajustados se necessário. </li></ol></li><li> Selecione um ponto estável e claro como um ponto de referência. Este ponto de referência pode ser qualquer ponto claro e pequenas estruturais que permanece inalterado e estável durante todo o experimento. Clique em &quot;Add Track&quot; para rastrear o ponto de referência do primeiro ao último frame e clique em &quot;Track End&quot;. Os dados aparecem na tabela de resultados automaticamente. </li><li> Acompanhar o rastreamento ea migração de neutrófilos, um por um: clique em &quot;Add Track&quot; para rastrear o celular de sua aparência no tecido ao seu desaparecimento em cada quadro e clique em &quot;Track End&quot; para finalizar e salvar os resultados no Microsoft Excel. </li></ol> 4. Análise dos Parâmetros recrutamento de neutrófilos <ol><li> Abra o arquivo de resultados no Microsoft Excel, e analisar os dados (se as mudanças de ponto de referência em análise). </li><li> Rastejando intraluminal <ol><li> Rastreamento de distância: a distância total da célula rasteja no lúmen do site inicial aderente ao site a transmigração ideal (m). </li><li> Crawling velocidade: rastreamento de distância / tempo (mM / min). </li></ol></li><li> Migração transendotelial <ol><li> Transmigração tempo: a partir do momento da célula começa a transmigrar através do endotélio ao tempo em que o corpo da célula toda é apenas fora da vênula e nenhum corpo celular pode ser visto no lúmen (min ou seg.) </li><li> Tempo de descolamento: a partir do momento em que o corpo da célula toda é apenas fora da vênula (imediatamente após a transmigração) para o ponto no tempo quando a célula perde o contato com a vênula (o retrai cauda) (min ou seg.) </li></ol></li><li> Quimiotaxia em tecido <ol><li> Distância de migração: a soma da distância da célula se move a partir do ponto inicial ao ponto final da migração no tecido (mm). </li><li> Velocidade de migração: a distância de migração no tecido / hora (mM / min). </li><li> Quimiotaxia distância: a soma de células a distância migra no eixo-x no tecido (mm). </li><li> Velocidade de quimiotaxia: quimiotaxia distância / tempo (mM / min). </li><li> Quimiotaxia índice: a taxa de dividir a distância quimiotaxia pela distância de migração no tecido. </li></ol></li></ol> 5. Resultados representativos: Embora bightfield microscopia intravital é utilizado para o estudo das interações leucócito-endotélio celular e não podem ser necessariamente para os neutrófilos, que confirmou que, por nossos estudos de histologia, mais de 95% das células recrutadas em neutrófilos tratados com músculos chemoattractant cremaster foram de fato neutrófilos . Neste relatório, usando neutrófilos seletivo quimioatrativos, apresentamos os procedimentos de monitoramento do recrutamento de neutrófilos in vivo. Especificamente, nós descrevemos um protocolo de rastreamento rastreamento de neutrófilos intraluminal, a migração transendotelial, e quimiotaxia em cremaster tecido muscular em ratos anestesiados com lapso de tempo de microscopia intravital de vídeo e ImageJ. O chemoattractant contendo gel agarose no músculo cremaster libera lentamente chemoattractant e permite um gradiente chemoattractant a ser estabelecida no tecido. Chemoattractant neutrófilos induz interações neutrófilos-célula endotelial em cremastérico vênulas pós-capilares em camundongos. Todo o experimento é visualizado sob um microscópio de campo claro vertical intravital com imagens de vídeo projetadas por uma câmera de vídeo de cor para um monitor de TV e gravado por um gravador de vídeo. Nós determinamos os neutrófilosrastejando intraluminal, a migração transendotelial e migração e quimiotaxia em tecido muscular em resposta à neutrófilos chemoattractant MIP-2 e WKYMVm preparada em gel de agarose (Figura 2). Descobrimos que MIP-2 (0,5 mM) e WKYMVm (0,1 mM) provocou rastejando intraluminal de neutrófilos em velocidade similar, a migração de neutrófilos transendotelial e desprendimento da vênula de comprimento comparável de tempo, a migração de neutrófilos e quimiotaxia no tecido muscular em quase a mesma velocidade e com índices similares quimiotaxia de neutrófilos (P&gt; 0,05, teste t de Student). <img src="/files/ftp_upload/3296/3296fig1.jpg" alt="Figura 1" /> Figura 1. A ilustração esquemática de um sistema de microscópio intravital. O mouse músculo cremaster é exteriorizado no pedestal visualização clara de cremaster bordo muscular no palco microscópio e superfused com 37 ° C-aquecido bicarbonato tampão salina. O microscópio na posição vertical é conectado com uma câmera de vídeo CCD de cor para brightfield microscopia intravital. A profunda monocromática CCD refrigerado a câmera digital também é conectado à porta microscópio de fluorescência para microscopia intravital, as imagens a partir do qual são diretamente processados ​​por um computador. <img src="/files/ftp_upload/3296/3296fig2.jpg" alt="Figura 2" /> Figura 2. Neutrófilos parâmetros recrutamento de brightfield microscopia intravital. Recrutamento de neutrófilos foi induzida pela liberação gradual de chemoattractant neutrófilos MIP-2 ou WKYMVm na preparação gel agarose colocados 350 mM adjacente à vênula postcapillary. Tempo decorrido dados de vídeo foram analisadas pelo ImageJ após o processamento da gravação de vídeo em tempo real do experimento. Neutrófilos rastejando intraluminal (A), o tempo de transmigração e tempo de descolamento (B), velocidade de migração e velocidade de quimiotaxia em tecido (C), e índice de quimiotaxia em cremaster muscular (D) foram determinados após a administração de MIP-2 ou agarose gel em WKYMVm cremaster muscular em camundongos C57BL / 6 (n = 3, # de células rastreado = 22 (em A e B) e 27 (em C e D), respectivamente, para MIP-2, e = 26 (em A e B) e 44 ( em C e D), respectivamente, para WKYMVm).

<ol>
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Microscopia intravital é a ferramenta essencial para revelar os mecanismos celulares e moleculares do recrutamento de leucócitos durante a inflamação. Visualização quantitativa para a determinação das interações leucócito-endotélio celular na microvasculatura de tecidos translúcido como o músculo cremaster e mesentério continua sendo o padrão ouro para a aplicação da técnica 1,5. A microscopia intravital convencionais brightfield tem muitas características únicas técnicas e os mecanismos...

Tabela: materiais, reagentes e equipamentos específicos <table border="1"><tr><td> Nome do reagente / equipamentos </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Tubos de polietileno, PI10 </td><td> Becton Dickinson </td><td> 427401 </td><td> ID 0,28 milímetros × 0,61 milímetros OD </td></tr><tr><td> Nanquim </td><td> Speedball Art </td><td> Super Preto </td><td> Pigmentos de carbono não 100% preto corantes </td></tr><tr><td> Cautério </td><td> Aaron Medical </td><td> AA03 </td><td></td></tr><tr><td> Xilazina </td><td> Bayer HealthCare, Bayer Inc. </td><td> DIN 02169592 </td><td></td></tr><tr><td> Cloridrato de cetamina </td><td> Bioniche Animal Health Canada, Inc. </td><td> DIN 01989529 </td><td></td></tr><tr><td> MIP-2 recombinante murino </td><td> R &amp; D Systems </td><td> 452-M2 </td><td></td></tr><tr><td> WKYMVm </td><td> Phoenix Pharmaceuticals, Inc. </td><td> 072-12 </td><td></td></tr><tr><td> Agarose </td><td> Invitrogen </td><td> 15510-027 </td><td> Ultrapura </td></tr><tr><td> Heparina </td><td> Sigma </td><td> H-3393 </td><td></td></tr><tr><td> Microscópio vertical </td><td> Olimpo </td><td> BX61WI </td><td></td></tr><tr><td> Câmera de vídeo 3CCD cor </td><td> SONY </td><td> DXC-990 </td><td></td></tr><tr><td> HD-DVD gravador de vídeo </td><td> LG Electronics Inc. </td><td> RH398H-M </td><td></td></tr><tr><td> Monitor de TV </td><td> LG Electronics Inc. </td><td> 22LG30 </td><td></td></tr><tr><td> Circulador de água </td><td> Thermo Scientific </td><td> DC10 HAAKE </td><td></td></tr><tr><td> Bomba peristáltica </td><td> Gilson; Pharmacia </td><td> Gilson MINIPULS 3; Pharmacia P-3 </td><td></td></tr><tr><td> Cremaster bordo muscular </td><td> Universidade de Saskatchewan </td><td></td><td> Home-made </td></tr></table>

tracking neutrophil intraluminal crawling, transendothelial migration and chemotaxis in tissue by intravital video microscopy

O recrutamento de leucócitos circulantes do fluxo de sangue para o tecido inflamado é um processo crucial e complexa de 1,2 inflamação. No vênulas pós-capilares do tecido inflamado, leucócitos inicialmente tether and roll na superfície luminal da parede venular. Rolando prisão leucócitos no endotélio e passam por adesão firme em resposta a quimiocina ou quimioatrativos outros na superfície venular. Muitos leucócitos aderentes mudar a partir do site inicial de adesão ao site extravasamento juncional no endotélio, um processo denominado intraluminal rastejando 3. Após rastreamento, os leucócitos movem-se através do endotélio (transmigração) e migrar no tecido extravascular em direção à fonte de chemoattractant (quimiotaxia) 4. Microscopia intravital é uma ferramenta poderosa para a visualização de interações leucócito-endotélio celular in vivo e revelando os mecanismos celulares e moleculares do recrutamento de leucócitos 2,5. Neste relatório, nós fornecemos uma descrição completa do uso de microscopia intravital brightfield de visualizar e determinar os processos detalhados de recrutamento de neutrófilos em camundongos músculo cremaster em resposta ao gradiente de uma chemoattractant neutrófilos. Para induzir o recrutamento de neutrófilos, um pequeno pedaço de gel de agarose (~ 1-3 mm de tamanho) contendo chemoattractant neutrófilos MIP-2 (CXCL2, uma quimiocina CXC) ou WKYMVm (Trp-Lys-Tyr-Val-Met-D, um análogo sintético de peptídeo bacteriana) é colocado no tecido muscular adjacente à vênula observado postcapillary. Com o tempo decorrido, fotografia, vídeo e computador software ImageJ, neutrófilos no endotélio rastejando intraluminal, a migração de neutrófilos e transendotelial a migração e quimiotaxia em tecido são visualizados e monitorados. Este protocolo permite a análise quantitativa confiável e de muitos parâmetros, tais como o recrutamento de neutrófilos velocidade rastejando intraluminal, o tempo de transmigração, tempo de descolamento, a velocidade de migração, a velocidade de quimiotaxia e índice de quimiotaxia em tecido. Nós demonstramos que a utilização deste protocolo, estes parâmetros recrutamento de neutrófilos pode ser determinado e de forma estável a locomoção única célula convenientemente monitorados in vivo.

Watch this Scientific Journal Video about Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy at JoVE.com

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

Nós descrevemos um protocolo de brightfield microscopia intravital para medir a dinâmica interações neutrófilos-célula endotelial durante o recrutamento de neutrófilos em resposta à fonte de um chemoattractant neutrófilos in vivo. Neutrófilos rastejando intraluminal, a migração transendotelial e quimiotaxia em tecido muscular do rato cremaster são visualizados com o tempo decorrido, fotografia, vídeo e rastreados com 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 ...

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JoVE Journal

Immunology and Infection

19.5K visualizações.  University of Saskatchewan. Nós descrevemos um protocolo de brightfield microscopia intravital para medir a dinâmica interações neutrófilos-célula endotelial durante o recrutamento de neutrófilos em resposta à fonte de um chemoattractant neutrófilos in vivo. Neutrófilos rastejando intraluminal, a migração transendotelial e quimiotaxia em tecido muscular do rato cremaster são visualizados com o tempo decorrido, fotografia, vídeo e rastreados com ImageJ.

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

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

Neutrófilos Traps Extracelular: Como Gerar e visualizá-los

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

Imunologia e Infecção

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Microscopia intravital do linfonodo inguinal

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Live-célula Microscopia vídeo de Fagocitose patógeno fúngico

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

In vitro Ensaio de cocultura para avaliar migração trans epitelial de neutrófilo induzida por patógenos

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.

We developed an in vitro model that mimics an important component of the acute inflammatory response during infection of the bladder with uropathogenic Escherichia coli. The transuroepithelial neutrophil migration assay enables quantitative assessment of human neutrophil migration across bladder epithelia, cultured on permeable supports, in response to bacterial infection or chemoattractant substances.

Avaliação quantitativa de neutrófilos humanos migração através de um Cultivadas bexiga Epitélio

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. ...

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 ...

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

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

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

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

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

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

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

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

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

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

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

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

Um método All-on-chip para análise rápida de quimiotaxis de neutrófilos diretamente de uma gota de sangue

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

In Vitro Formação canina neutrófilos extracelular armadilha: Análise dinâmica e quantitativa por microscopia de fluorescência

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Methods Article

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