Gostaríamos de agradecer Ying Yu para APC Min / + camundongos genotipagem. Este estudo foi apoiado por fundos do INSERM e subvenções (CA057621 e AI053194) do National Institutes of Health.

NOTA: Todos os experimentos com animais foram realizados de acordo com procedimentos aprovados pelo Comitê de Cuidados e Uso Animal Institucional (IACUC), UCSF. Todos os experimentos com imagens eram procedimentos não-sobrevivência e os animais foram sacrificados imediatamente após o final da aquisição da imagem. 1. geração de camundongos NOTA: Apc Min / +, ratinhos portadores de uma mutação no gene APC, desenvolvem espontaneamente 50-100 adenomas do intestino delgado. <ol><li> Cruz Apc Min / + ratinhos com a linha de ACTB-ECFP, ECFP que expressa sob o promotor de actina, e ainda com a linha de c-fms-EGFP, que expressa EGFP sob o promotor de c-fms para detectar as células mielóides. Animais heterozigóticos para ACTB-ECFP e c-fms-EGFP são utilizados para detectar a fluorescência. </li><li> Use camundongos em 3-4 meses de idade a imagem adenomas claramente detectáveis. </li></ol> 2: Preparação de Materiais antes da cirurgia NOTA: Os detalhes do microscópio set-up ter sido anteriormente descrito 6,7. <ol><li> Microscópio <ol><li> Ative o cobertor térmico. Ligue o laser de argônio para 488 nm de excitação e de estado sólido de 405 nm, 561 nm e 640 nm lasers. Ligue o microscópio, a unidade de controlo de disco giratório, a AOTF, a unidade de controlo do laser, e do controlador da câmara. </li><li> Abra o obturador microscópio, que transforma o LED vermelho. Ligue o computador e câmera de software. </li></ol></li><li> Plataforma de Imagem <ol><li> Certifique-se de que a inserção palco é limpo. Caso contrário, limpe com água e sabão e depois seque. </li><li> Tome duas lamelas e usar fita laboratório para fixá-los na inserção. Coloque a inserção no palco e limpe-o com algodão embebido em álcool. </li><li> Usando fita laboratório, anexar a manta de aquecimento para o lado direito do palco. </li><li> Verificar a integridade da membrana de borracha sobre o cone de nariz e que se mudam necessary. Posição e fixar o cone do nariz para entrega anestesia isoflurano ao animal usando gaze e fita de laboratório. </li></ol></li><li> O isoflurano anestesia Sistema <ol><li> Encher o tanque de isoflurano. </li><li> Ligar o vácuo e ajustar para 1,2 L / min. </li><li> Adicione água destilada até o nebulizador e ligar o nebulizador para o sistema de isoflurano. </li><li> Ligue o analisador de oxigênio e conectá-lo ao sistema de isoflurano. Ligue o tanque de oxigênio e garantir que o analisador de oxigênio mostra 100%. Ajustar o fluxo de O 2 a 0,2 L / min. </li><li> Ligue o tanque de nitrogênio e ajustar o fluxo de 0,8 L / min. Verifique se o analisador de oxigênio mostra 21%. </li></ol></li><li> Preparação de Ferramentas de Cirurgia e Plataforma <ol><li> Limpe duas pinças e tesouras de dissecação com água e sabão. </li><li> Ligue o esterilizador talão quente e deixá-lo chegar a 250 ° C. Esterilizar os instrumentos cirúrgicos para 30 seg. Deixe-os esfriar. </li><li> Coloque uma imersão laboratório no banco cirurgia. </li><li> Use uma tampa de isopor como uma plataforma cirúrgica. Cubra com um pedaço de laboratório de imersão. Prepare um segundo pedaço de laboratório giratória para mudar após o barbear o mouse. </li><li> Coloque o cone do nariz com a linha de anestesia na tampa de isopor coberto, e anexá-lo para a tampa de isopor (não no laboratório de imersão) com alguma fita e usar duas agulhas em ambos os lados do cone do nariz para mantê-lo no lugar. </li><li> Montar betadine, toalhetes com álcool, gaze estéril, super cola, lâminas de microscópio, barbeador elétrico, e quatro pedaços de fita de laboratório. </li></ol></li></ol> 3 Preparação de Injeções <ol><li> Sob o capô, preparar uma seringa de insulina (28 G x ½ cm), com 7 ul de estoque AF647 conjugado Ly-6G (Gr1) anticorpo (1 mg / ml) em 100 ul de 2000 kDa rodamina-dextrano (4 mg / ml ) para injecção retro-orbital. Injeção retro-orbital é recomendado na APC min / + camundongos desde anemia que se desenvolve nesses animais faz com que o difficu veias da caudalt para detectar através da pele. </li><li> Prepara-se uma segunda seringa de insulina com 1 mg / kg de atropina diluído em 150 ul de PBS por via subcutânea (sc) de injecção antes de imagem para amortecer movimentos peristálticos do intestino. </li></ol> 4 Preparação do Intestino for Imaging <ol><li> Verifique se a linha de anestesia para a câmara de indução está aberto, e as linhas para a mesa cirúrgica e microscópio estão fechados. </li><li> Transferir o animal à câmara de anestesia. Fechar a câmara de anestesia e ligar o isoflurano a 4% (com O 2 21%). </li><li> Quando o mouse respira profundamente e lentamente (após 5-6 min), transferi-lo para o estágio cirúrgico no seu flanco e injetar a solução de anticorpos Gr1 e / ou solução de dextrano retro-orbital. </li><li> Abrir o tubo de anestesia ligada à plataforma cirúrgica. Feche a linha para a câmara de anestesia e coloque o mouse sobre a sua volta com seu nariz no cone anestesia. </li><li> Reduzir o isofluranoconcentração de 4% a 2,5%. </li><li> Verifique se o mouse é bem anestesiados por beliscar a sua pata e teste do reflexo corneal. </li><li> Adicionar oftálmica pomada lubrificante nos olhos para evitar a secura sob anestesia. </li><li> Prenda os membros do mouse para o estágio cirúrgico, acrescentando fita laboratório. </li><li> Remover os pêlos da superfície ventral usando um barbeador elétrico. Também remova o cabelo da pata traseira esquerda para posicionar o oxímetro antes da aquisição. Retire a imersão laboratório a partir da fase cirúrgica e substituir por uma limpa para evitar a contaminação de cabelo. </li><li> Desinfetar a superfície ventral com algodão embebido em álcool e betadine. </li><li> Injectar a solução de atropina (de 3,2) sc em um flanco do mouse. </li><li> Adicione de 1 cm ventral incisão na linha média através da pele e do peritoneu com uma tesoura e por um par de pinças. Retire cuidadosamente 3-4 cm de intestino e identificar um ou vários tumores de imagem. </li><li> Tome um copo de MICRoscope corrediça e a posição de uma linha de assinante do intestino em que com um tumor no topo. </li><li> Adicione um pouco de super cola para alguns locais ao longo do intestino para anexá-lo ao slide. Esperar um minuto para a cola secar. Usar um marcador para desenhar uma linha sobre a lâmina apontando para o tumor de modo a facilitar a sua localização com o confocal. </li></ol> 5. posicionar o mouse no Palco e Preparação para Aquisição de Imagem <ol><li> Retire a fita de laboratório de membros. </li><li> Abrir o tubo de anestesia para a fase de microscópio e fechar a uma plataforma para o cirúrgico. </li><li> Transferir o mouse com cuidado para o estágio do microscópio com o lado ventral para baixo e seu nariz no nariz do avião. Prenda a linha de anestesia com alguma fita laboratório. </li><li> Re-posicione o mouse e / ou o slide, a fim de ter o intestino no centro de uma das janelas caiu de cobertura. Gentilmente fita para baixo o slide com fita laboratório. </li><li> Fixe a sonda oxímetro no membro esquerdo do mouse parasiga seu coração e níveis de taxas e de saturação de oxigênio arterial respiratórias. </li><li> Cubra o mouse com a manta de aquecimento para evitar a hipotermia. </li><li> Diminuir a concentração de isoflurano de 2,5% para 1-1,5% para exames. </li></ol> 6 Aquisição de imagens usando o μManager Software <ol><li> Aumente a velocidade CSUX a 2.500 rpm para melhorar a qualidade da imagem nas definições de configuração. </li><li> Use o menu suspenso para escolher Objetivo 10X 0,5 NA ou 20X 0,75 NA Fluar da lente objetiva. </li><li> Escolha o laser de 405 nm menu dropdown Color. </li><li> Clique na Live e ajustar o foco com o microscópio. </li><li> Clique em Auto para ajustar automaticamente intensidades máximas e mínimas de pixel da imagem de exibição com base nos valores de pixels extremos da imagem. Um histograma de intensidade dos pixels da imagem da tela é apresentada no gráfico na parte inferior da janela principal. </li><li> Ajuste a exposição da câmera na janela do Painel de Controle Piper poralterando o número de relógios (um relógio é 33,333 ms). </li><li> Se o sinal estiver muito brilhante com a menor exposição, diminuir a intensidade do laser, usando a unidade de controle do microscópio. </li><li> Verifique a intensidade do sinal para 488 nm, 561 nm e 640 nm linhas de laser. Ajustar a intensidade do laser com a sua própria unidade de controlo do laser de (488 nm e 561 nm) ou a unidade de controlo do microscópio. </li><li> Na janela de multi D Aquisição, marque a caixa pontos Tempo e digitar o número de pontos de tempo e intervalo de tempo desejado. </li><li> Verifique a caixa de Z-pilhas, escolha &quot;Z relativa&quot; e definir Z-start e Z-final em relação à posição atual. NOTA: Para 10X ou 20X objetivo, comece com 3 planos Z, 8 m ou 4 mm para além respectivamente. </li><li> Marque a caixa Várias posições (XY) para geração de imagens de vários locais, em seguida, clique em Editar lista de posição, marcar 4-6 posições diferentes. </li><li> Verifique a caixa de Canais e adicionar canais de Novo e selecione lasers linhas do menu suspenso e tipe em exposição para cada canal (em múltiplos de 33,333 ms). </li><li> Verifique Salvar imagens na janela &quot;aquisição multi D&quot;. </li><li> Selecione uma pasta e todas as imagens adquiridas serão automaticamente salvos nesta pasta com o prefixo do nome dado. NOTA: Cada aquisição contínua será salvo em uma subpasta separada, como arquivos TIFF individuais para cada fatia Z em cada cor em cada ponto de tempo. </li><li> Salve todas as configurações clicando em Salvar configurações na janela de multi D Aquisição. </li><li> Clique em Obter na janela de aquisição do Multi-dimensional para começar a aquisição. </li><li> Se a sonda oxímetro não é usado ou pára de funcionar bem (quando pulso não é estável ou difíceis de detectar), verificar a eficiência da anestesia a cada 15 minutos durante o procedimento de imagem por beliscar a pata e verificar o reflexo corneal. Ajuste as anestesia se o mouse é sensível a estes estímulos. </li><li> Após a aquisição, a eutanásia o mouse, aumentando a concent isofluranoração de 5%. Quando há movimentos respiratórios são detectáveis, retire o mouse do palco e realizar um deslocamento cervical. </li></ol> 7 Análise de imagens usando o Imaris Software <ol><li> Conversão para .IMS Arquivos (Recurso Figura 1A) <ol><li> Abra o software da câmera, clique em Arquivo, em seguida, o conversor de grupo. </li><li> Clique em Adicionar arquivos e escolha o primeiro arquivo da primeira posição. Repita o procedimento para cada posição. </li><li> Para cada arquivo carregado, clique em Configurações e verifique se as configurações de &quot;t&quot; para o tempo, &quot;c&quot; para os canais e &quot;z&quot; para Z-stacks aparecem nessa ordem. </li><li> Salve na pasta desejada. Navegar e criar um novo arquivo convertido. </li><li> Clique em Set para todos e Start. </li></ol></li><li> Ajustes (Recurso Figura 1B) <ol><li> Vá para a pasta convertido específico e abrir o primeiro arquivo. </li><li> Na janela de Ajuste Display, ajustar o fundosinal de corte, modificando o número mínimo para cada canal, se necessário. </li><li> Se necessário, ajustar a intensidade do sinal através da modificação do número máx (quanto menor o número máximo, maior é a intensidade do sinal). NOTA: Os ajustes do sinal de intensidade / contraste permitir o destaque de um comportamento de célula ou um compartimento para ter uma visualização mais agradável. Isso não muda o resultado em si. </li><li> Clique em Editar e Imagem em Propriedades. </li><li> Mudar as x, y, e z (valores para uma objectiva 10X, x e y = 0,712, z = 8 um, por uma objectiva 20X: x e y = 0,36, z = 4 mm). </li><li> Clique em Todos os Eqüidistante para ajustar pontos de tempo. Adicione a data de início ea hora de início da aquisição. Adicione a data final e hora final da aquisição (Figura 1C Suplementar). </li><li> Clique no botão Play para assistir ao filme. </li><li> Clique em Editar e excluir pontos de tempo para apagar as imagens borradas. </li><li> Para juntar duas aquisições separadas together em um filme, ir para o último instante do primeiro filme e clique em Editar&gt; Adicionar pontos de tempo para adicionar o segundo arquivo. </li></ol></li><li> Salvando como um filme (Figura 1D Suplementar) <ol><li> Clique na Record, escolha a extensão mov e um factor de compressão de 0. </li><li> Clique em Salvar. </li></ol></li></ol>

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The authors have nothing to disclose.

Neste trabalho, um protocolo detalhado é descrito por disco giratório de imagem confocal da dinâmica de células mielóides em tumores intestinais por várias horas em um animal vivo, fotografada do lado da serosa do intestino. Para evitar a inflamação e a ter condições fisiológicas óptimas, a imagiologia do intestino tem que ser feito no órgão intacto. No entanto, a imagem do lado da serosa do intestino é difícil, uma vez que a luz tem de passar através de diferentes camadas d...

Evidência esmagadora demonstra agora que o microambiente do tumor, consistindo em populações celulares heterogéneas, incluindo fibroblastos, células endoteliais, células imunitárias e inflamatórias, matriz extracelular e factores solúveis, desempenha um papel crucial na iniciação e progressão de tumores sólidos, contribuindo para quase todos características de câncer 1. Com efeito, durante a progressão do tumor, há interações dinâmicas constantes entre as células cancerosas transformadas e células do estroma que evoluem para gerar um microambiente favorável à malignidade 2. Entre as células do sistema imunológico que se infiltram no microambiente do tumor, as células mielóides são os mais abundantes 3. Consistindo de macrófagos associados a tumores (TAM), células supressoras derivadas de mielóides (MDSCs), células dendríticas (DC) e de neutrófilos (PMNs), as células mielóides são recrutadas a partir da medula óssea e infiltrar progressivamente tumores, libertando citocinas, factores de crescimento e proteases que pode promovercrescimento do tumor e se espalhar 4. A interferência entre células cancerosas e células mielóides é complexo, mas dinâmico. Assim, a compreensão da natureza de suas interações é crucial para determinar por que essas células promovem a progressão do câncer, em vez de participar de uma resposta imune anti-tumor, e pode ajudar a encontrar novos alvos para controlá-lo. A observação direta por microscopia intravital fornece informações sobre a dinâmica de células dentro dos tecidos de camundongos ao vivo 5. A quatro cores, multi-região, disco giratório com lentes micro-sistema confocal foi projetado para estudar células estromais dentro de tumores mamários 6. Esta abordagem permite a imagiologia contínua a longo prazo e inclui várias vantagens, tais como: (a) de aquisição de imagens rápidas para minimizar artefactos de movimento, (b), a anestesia de longo prazo, (c) quatro aquisição de cor a seguir diferentes tipos de células, (d) marcação fluorescente de diferentes componentes tumorais, e (e) a observação de diferentes microambientes tumorais sagacidadehin o mesmo mouse para evitar mouse para variabilidade rato 7-9. Com esta tecnologia, os diferentes comportamentos de células têm sido relatados no vírus do tumor mamário de modelo (MMTV) polioma meio conduzido pelo promotor T oncogene (Pymt) que mostra as fases progressivas da tumorigénese. Os linfócitos T reguladoras (Treg, visualizadas pelo transgene Foxp3 EGFP) migram preferencialmente na proximidade dos vasos sanguíneos que o DCS (CD11c-DTR-EGFP), fibroblastos associada a carcinoma (FSP1 + / + -EGFP) e células mielóides (c-fms -EGFP) exibem maior mobilidade na periferia do tumor do que no interior da massa do tumor. Na condição de hipóxia sistémica aguda, as células migraram de forma diferente: Tregs parar a migração em contraste com as células mielóides que continuam a mover-se 6. Além disso, no mesmo modelo de rato, demonstrou-se que as alterações de sensibilidade com doxorrubicina fase do tumor, a distribuição da droga está relacionada com a resposta a fármacos, e doxorrubicinatratamento conduz ao recrutamento dependente de CCR2 de células mielóides ao tumores. Assim, imagens ao vivo também pode ser usado para obter insights sobre as respostas de drogas no local e na biologia do chemoresistance 10,11. Polipose adenomatosa coli (APC) mutações genéticas ocorrem geralmente em adenomas colorretais humanos e carcinomas 12 e mutação de uma única cópia dos resultados gene APC em polipose adenomatosa familiar (FAP), o que confere um risco extremamente elevado para câncer de cólon 13. A estirpe de ratinhos Apc Min / + carrega uma mutação no códon 850 truncamento do gene APC e desenvolve espontaneamente várias adenomas intestinais em todo o intestino delgado de 14 16. Imagens intravital longo prazo do intestino é difícil por causa da capacidade de invasão do procedimento, uma vez que a abertura da cavidade peritoneal, é necessária para o acesso ao intestino. Imagens ao vivo de curto prazo ruaudies ter sido previamente publicada no intestino saudável 17,18, mas a observação direta de longo prazo de tumores intestinais não foi relatado. Um procedimento cirúrgico foi concebido e aperfeiçoado para visualizar tumores através da superfície serosa do intestino, usando o sistema de microscopia intravital fiação disco previamente utilizado para tumores mamários 6,10 imagem. Neste artigo, é descrito um protocolo que permite que se siga o comportamento de células mielóides no interior dos tumores do intestino delgado, utilizando Apc Min / + mice.

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			<td>2020</td>
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			<td height="42" style="height:42px;width:216px;">cfms-EGFP mice</td>
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			<td height="42" style="height:42px;width:216px;">2,000 kDa Dextran, rhodamine-conjugated</td>
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			<td>24x50-1</td>
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			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:141px;">12-550-15</td>
			<td>25x75x1mm</td>
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real-time imaging of myeloid cells dynamics in apcmin/+ intestinal tumors by spinning disk confocal microscopy

Células mielóides são as células do sistema imunológico mais abundantes e dentro de tumores têm sido mostrados para promover a progressão do tumor. Técnicas de imagem intravital modernos permitem a observação do comportamento celular ao vivo dentro do órgão, mas pode ser um desafio em alguns tipos de câncer devido ao órgão e tumor acessibilidade, tais como intestino. A observação direta de tumores intestinais não foi previamente relatado. Um procedimento cirúrgico descrito aqui permite a observação direta da dinâmica de células mielóides dentro dos tumores intestinais em ratos vivos, utilizando camundongos transgênicos repórter fluorescente e traçadores injectáveis ​​ou anticorpos. Para este propósito, um de quatro cores, multi-região, com lentes de micro-disco giratório microscópio confocal que permite imagens contínuo a longo prazo com a aquisição de imagem foi usada rápida. Apc Min / + ratinhos que desenvolvem adenomas múltiplos do intestino delgado são cruzados com ratos c-fms-EGFP para visualizar as células mielóides e com camundongos ACTB-ECFPpara visualizar as células epiteliais do intestino das criptas. Procedimentos para a marcação de diferentes componentes, tais como tumores, vasos sanguíneos e neutrófilos, e o processo para o posicionamento do tumor para a imagiologia por meio a superfície serosa também são descritos. Filmes time-lapse compilados a partir de várias horas de imagens permitem que a análise do comportamento de células mielóides in situ no microambiente intestinal.

Usando fiação de microscopia confocal de disco, tecidos não tumorais e tumorais no intestino delgado de Apc Min / +; ACTB-ECFP; ratinhos c-fms-ECFP pode ser visualizada a partir da superfície serosa. Depois de imagem, software da câmera é usada para analisar e ajustar a aquisição (Figura Complementar 1). Após a administração intravenosa (iv) injecção de 2.000 kDa fluorescente rodamina-dextrano e um anticorpo conjugado Ly-6G 647, vasos sanguíneos e PMN ...

Watch this Scientific Journal Video about Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy at JoVE.com

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 ...

real-time-imaging-myeloid-cells-dynamics-apcmin-intestinal-tumors

Research

JoVE Journal

Immunology and Infection

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

9.6K visualizações.  INSERM U661, Functional Genomic Institute. 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. 

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

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

Microscopia intravital do Baço: Análise Quantitativa da Mobilidade Parasite e do fluxo sanguíneo

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Imunologia e Infecção

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

A técnica de microscopia TIRF para imagens em tempo real, simultâneo da TCR e seus associados proteínas sinalizadoras

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

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 ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

Avaliar a atividade anti-fúngica isolada de macrófagos alveolares por microscopia confocal

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

A linfa mesentérica Duct canulada Rat Modelo: Aplicação para a Avaliação de Intestinal Linfática transporte da droga

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Indução de Murino inflamação intestinal por transferência adotiva de Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alta</sup&gt; Células T em imunodeficientes Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

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

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

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

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

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

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

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

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

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

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

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

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.