Os autores gostariam de agradecer ao Dr. Alain Trautmann que nos encorajou a realizar cortes de linfonodos. Este trabalho foi financiado em parte por concessões do Nationale Ligue Contre le Cancer, a Fondation pour la Recherche Médicale en France ea Associação pour la recherche sur le Cancer.

1. Preparação de fatias de linfonodos <ol><li> Prepare a 4% de baixo ponto de fusão de solução de agarose para a incorporação. Diluir agarose em PBS e microondas esta solução. Quando a agarose é completamente dissolvido, manter a solução a 37 ° C até que esteja pronto para uso. </li><li> Prepare um 6 bem-placa de cultura de tecidos. Adicionar 1,1 ml meio de cultura RPMI completo por poço, e inserir um filtro organotípicas em cada poço. Coloque a placa de seis poços a 4 ° C até que esteja pronto para transferir fatias. </li><li> Coloque arruelas de aço inoxidável, com diâmetros interno de 4 mm, em um prato de plástico cheio de meio RPMI completo. Arruelas serão mais usados ​​para concentrar as células na fatia cortada vibratome. </li><li> Sacrificar o rato, em conformidade com as normas de bem-estar animal local. Remova cuidadosamente os gânglios linfáticos periférico do tecido circundante e coloque-os em um prato de plástico contendo gelada PBS. Cuidados devem ser tomados para não danificar a estrutura dos linfonodos, como estes órgãos são muito macios. </li><li> Despeje a 4%-agarose gel em um prato de plástico de 35 mm e delicadamente os nós de transferência para o gel. Deixe o gel agarose em gelo por 5 min para endurecer. </li><li> Remover o bloco do prato e cortar o agar, a fim de deixar 3-5 mm de gel em torno de cada nó de linfa. </li><li> Anexar os nós embutido no disco exemplar do vibratome com non-toxic cola de tecido. Instalar o disco de amostra na bandeja cheia de gelado PBS. </li><li> Seção agar-embedded tecido em 320 mM espessura com a velocidade vibratome conjunto em uma série lenta (0,3 mm / s) ea freqüência de vibração em um conjunto de gama média (1,5 mm). </li><li> Transferir cuidadosamente fatias linfonodo como eles estão sendo cortados para a cultura insere organotípicas usando uma pinça fina. Coloque 3 fatias em cada inserção. Tenha muito cuidado como fatias pode ser facilmente danificada. Um linfonodo periférico típico renderá 5 seções quando cortado a 320 mM. Descartar as fatias e sobrenome, como eles só contêm tecido superficial. </li><li> Coloque anilhas de aço inox em cada fatia individual. Certifique-se de arruelas são bem posicionado na agarose em torno do tecido. </li><li> Incubar a placa de cultura a 37 ° C em 5% incubadora de CO 2 umidificado até que esteja pronto para as células T placa. </li></ol> 2. Isolamento de células T dos linfonodos do mouse <ol><li> Isolar as células T de acordo com o artigo JOVE anteriormente publicados 3. </li><li> Utilização de células T imediatamente e prosseguir para a próxima seção. Caso contrário, os linfócitos podem ser cultivadas durante a noite em completa meio RPMI suplementado com 10 ng / ml de IL-7. </li></ol> 3. Rotulagem dos isolaram células T <ol><li> Lavar as células em HBSS. Ressuspender-los no mesmo tampão de 10 x 10 6 por ml. </li><li> Adicionar o mesmo volume de HBSS contendo 1 mM celular Rastreador verde CMFDA dando uma concentração final de 0,5 mM. </li><li> Incubar a suspensão de células a 37 ° C durante 5 min. Lave as células duas vezes com RPMI médio completo. </li><li> Ressuspender as células em uma concentração final de 10 x 10 6 por ml em meio RPMI completo. Estas células marcadas serão semeados em fatias. </li></ol> Outros corantes fluorescentes que CMFDA pode ser usado também, mas tenha em mente que estas moléculas são potencialmente tóxicos acima de determinadas concentrações. 4. Chapeamento rotulados células T em fatias linfonodo <ol><li> Remova o meio de excesso contido na parte interna da máquina de lavar com uma pipeta. Não permita que o tecido se torne seca; trabalhar rapidamente durante esta etapa. </li><li> Suavemente dispensar 10-20 mL células T rotulados que corresponde a 1-2 x 10 5 células na parte interna da máquina de lavar. É preciso ter cuidado para não tocar o tecido com a ponta da pipeta. Certifique-se que a gota de suspensão de células permanece no local. </li><li> Coloque as fatias com células em estufa a 37 ° C e 5% CO 2 por pelo menos 30 min para permitir que os linfócitos para migrar para o tecido. </li></ol> 5. Células T de imagens dentro de fatias de linfonodo Neste vídeo-artigo, que as células T imagem fluorescentes em fatias de linfonodo com um microscópio de campo amplo invertido e um microscópio confocal vertical. <ol><li> Definir a temperatura a 37 ° C. Nossos microscópios estão equipados com câmaras de temperatura controlada. </li><li> Instalar um sistema de perfusão permitindo a perfusão contínua da fatia de linfonodo com oxigenada (5% CO 2, 95% O 2) fenol meio RPMI red-free. No nosso sistema, media perfundidos entra na câmara de imagem de um lado pela gravidade. O meio é aspirado com uma bomba ligada a um frasco de coleta de resíduos. Definir a taxa de fluxo de mídia para 1 ml / min. </li><li> Com uma pinça fina, delicadamente retire a fatia do prato 6-bem e mergulhe a preparação em meio RPMI oxigenado aquecido por alguns segundos para se livrar de células fluorescentes não infiltradas no tecido. </li></ol> Imaging células T com um microscópio de campo amplo invertida <ol start="4"><li> Coloque a fatia de cabeça para baixo em uma câmara de imagem feito por encomenda, que consiste em fios de nylon colados na parte interna de um prato fundo de vidro. Nesta câmara, os fios de nylon elevar a fatia do vidro e permitem que a solução oxigenada para difundir no tecido. Isto é necessário como T migração celular em linfonodos é fortemente dependente de oxigênio. Segura a fatia acrescentando por cima em uma arruela de aço inoxidável. Nessas condições, a fatia está alguns mícrons acima do fundo do prato, o que facilita a renovação da solução de perfusão. </li><li> Para capturar um grande campo de visão (800 x 800 mM, aproximadamente um terço da superfície do nó fatia), recolher imagens usando uma lente objetiva de 10x e seco. Nós descobrimos que a Nikon 10x S fluor 0,5 NA foi mais adequado para nossas análises. </li></ol> Uma experiência de imagens típicas de lapso de tempo captura 5 aviões óptico abrangendo uma profundidade total de 50 mm na dimensão axial (z). Células T fluorescentes são normalmente fotografada em intervalos que variam 10 a 30 segundos durante 10 a 20 min. Células T de imagem com um microscópio confocal vertical O microscópio confocal utilizado neste protocolo é um SP5 Leica equipada com uma objetiva de imersão de água 20x (Olympus, 20x/0.95 NA). <ol start="6"><li> Segura a fatia para o palco de imagens do microscópio. Nota: Nós geralmente lugar uma máquina de lavar na preparação para imobilizá-lo. </li><li> Definir uma sessão de imagem através da recolha de uma série de imagens ao longo do eixo z. Para as células T de imagem em uma estrutura de tecido intacto, a posição de início é geralmente definida em 5 a 10 mM abaixo da célula T primeiro rotulados. </li></ol> 6. Resultados representativos Seguindo este vídeo protocolo você deve esperar para visualizar um grande número de células T fluorescentes acumuladas na zona T do nó, um fenômeno que normalmente ocorre nesse ambiente específico in vivo (Figura 1). Em particular, as células T devem ser excluídas das zonas de células B, que são geralmente logo abaixo da cápsula. A experiência bem sucedida também irá resultar em células T recrutados para o tecido (Figura 2), exibindo um comportamento altamente móveis (Figura 3) consistentes com os resultados publicados obtidos nos gânglios linfáticos intactos 4. Em média, a velocidade média de células T individuais dentro fatias devem ser cerca de 10 mM / min (Figura 4). <img src="/files/ftp_upload/3054/3054fig1.jpg" alt="Figura 1"/> Figura 1. As células T se acumulam em uma fatia de linfonodos. Células fluorescente marcado T (CMFDA, verde) foram adicionados a um nó de linfa fatia min 30 antes de aquisição de imagem (imagem acima). A imagem é a projeção máxima de 5 imagens que mede 50 mm na direção z. A imagem de campo claro é mostrado na parte inferior. Imagens foram capturadas com um microscópio de campo amplo. <img src="/files/ftp_upload/3054/3054fig2.jpg" alt="Figura 2"/> Figura 2. As células T são recrutados para uma fatia de linfonodos. Células fluorescente marcado T (CMFDA, verde) foram adicionados a um nó de linfa fatia min 30 antes de aquisição de imagem usando um microscópio confocal. Imagens foram capturadas na superfície de corte (figura superior) e 40 mM abaixo (foto inferior). <img src="/files/ftp_upload/3054/3054fig3.jpg" alt="Figura 3"/> Figura 3. As células T são altamente móveis dentro de uma fatia de linfonodos. Fluorescente marcado células T foram fotografadas durante 12 min usando um microscópio de campo amplo. Trajetórias de células T individuais são exibidos como codificados por cores para representar faixas deslocamentos crescentes de azul (baixo células móveis) ao vermelho (alto células móveis). Faixas foram calculados utilizando software Imaris. A linha branca segue a borda do nó, enquanto a elipse tracejada delimita a zona de células B putativa. <img src="/files/ftp_upload/3054/3054fig4.jpg" alt="Figura 4"/> Figura 4. Dentro de uma fatia de linfonodos, a velocidade de células T é cerca de 10 mM / min. As velocidades foram calculadas utilizando software Imaris de faixas representadas na Figura 3.

<ol>
	<li>Asperti-Boursin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCR7+ligands+control+basal+T+cell+motility+within+lymph+node+slices+in+a+phosphoinositide+3-kinase-independent+manner.">CCR7 ligands control basal T cell motility within lymph node slices in a phosphoinositide 3-kinase-independent manner.</a> J Exp Med. 204, 1167-1167 (2007).</li><li>Bhakta, N. R., Oh, D. Y., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+oscillations+regulate+thymocyte+motility+during+positive+selection+in+the+three-dimensional+thymic+environment.">Calcium oscillations regulate thymocyte motility during positive selection in the three-dimensional thymic environment.</a> Nat Immunol. 6, 143-143 (2005).</li><li>Matheu, M. P., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+CD4++T+cells+from+mouse+lymph+nodes+using+Miltenyi+MACS+purification.">Isolation of CD4+ T cells from mouse lymph nodes using Miltenyi MACS purification.</a> J Vis Exp. , (2007).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1869 (2002).</li><li>Ehrlich, L. I., Oh, D. Y., Weissman, I. L., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+contribution+of+chemotaxis+and+substrate+restriction+to+segregation+of+immature+and+mature+thymocytes.">Differential contribution of chemotaxis and substrate restriction to segregation of immature and mature thymocytes.</a> Immunity. 31, 986-986 (2009).</li><li>Gollmer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL21+mediates+CD4++T-cell+costimulation+via+a+DOCK2/Rac-dependent+pathway.">CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway.</a> Blood. 114, 580-580 (2009).</li></ol>

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Nós descrevemos uma técnica simples rápida e robusta para a geração de fatias de linfonodo, que são usados ​​para investigar o comportamento de células T introduziu. Nos últimos anos, este método tem sido aplicado com sucesso usando timo e linfonodo fatias para identificar os fatores extracelulares controlar o posicionamento de células T e mobilidade 1,5. Também tem sido usado para medir Ca 2 + respostas em timócitos durante a seleção positiva e em células T após o reconhecimento antígeno 2,6. O ensaio fatia overlay apresenta vantagens e limitações que merecem ser discutidas. De notar que este sistema permite o acesso ao tecido, útil se a pessoa precisa para manipular fatias farmacologicamente, a fim de interferir com o controle molecular de migração celular. Subseqüente ao de imagem, as fatias podem ser processados ​​por imuno-histoquímica para coletar informações sobre as estruturas que foram retratadas. Além disso, a observação pode ser feita com um microscópio de fluorescência tradicional campo amplo. Embora a resolução não é tão bom quanto com uma ou um microscópio confocal de dois fótons e que fototoxicidade é, em princípio, mais grave, com um fóton do que com a microscopia de dois fótons, ele tem as vantagens de simplicidade, um menor custo e maior escolha de comprimentos de onda de excitação. A imagem de células T em um linfonodo intactas requer a injeção intravenosa de linfócitos marcados que, posteriormente, o lar de órgãos linfóides. Como já mostrado anteriormente 1, o ensaio fatia é perfeitamente compatível com a experiência de transferência adotiva. No entanto, o período de tempo necessário para as células T a casa em gânglios linfáticos pode complicar o uso de corantes fluorescentes que têm uma tendência a vazar para fora das células ao longo do tempo. Este é o caso do Ca 2 + corante fura-2. Com o recrutamento rápido (&lt;30 min) de células T no tecido, o ensaio fatia fornece uma oportunidade de usar tais corantes. Finalmente, este método tem a grande vantagem de analisar as funções das células T em vários tecidos humanos mantidos vivos. Este sistema experimental apresenta também limitações que precisam ser mantidos em mente. Danos associados com o corte pode afetar o funcionamento das células T, especialmente na região superficial do tecido perto da superfície de corte. A fim de avaliar a morte celular dentro da fatia, temos utilizado a corante fluorescente verde SYTOX que penetra as células com membrana plasmática comprometida. Nossos experimentos revelam que cerca de 20% do total de células nodais, principalmente localizados na região superficial do tecido, foram fluorescente etiquetado com o corante nuclear. Outra preocupação com o potencial do ensaio é a capacidade de reter as fatias importantes fatores solúveis incluindo quimiocinas. Embora, não temos nenhuma indicação de que nossos dados foram afetados por problemas tais como as células T exibir uma boa mobilidade dentro da fatia, gostaríamos de salientar a importância das células T de imagens em regiões saudáveis ​​localizado em várias dezenas de microns da superfície de corte. Considerando que, isso poderia ser feito com microscópios tradicionais (campo amplo ou confocal), como neste protocolo, é provável que a linfa preparação fatia nó combinado com dois fótons de imagem vai aumentar a resolução espacial de profundidade e reduzir a fototoxicidade.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Microscópio confocal </td><td> Leica </td><td> SP5 </td><td></td></tr><tr><td> Vibratome </td><td> Leica </td><td> VT1200S </td><td></td></tr><tr><td> Pinça fina </td><td> Precision Instruments mundo </td><td> 14142 </td><td></td></tr><tr><td> 30 milímetros insere Cultura </td><td> Millipore </td><td> PICM0RG50 </td><td></td></tr><tr><td> RPMI </td><td> Invitrogen </td><td> 61870010 </td><td> RPMI completo e médio é feita pela adição de 10% inativado pelo calor de soro fetal bovino e penicilina / estreptomicina </td></tr><tr><td> Solução de Hanks salina balanceada (HBSS) </td><td> Invitrogen </td><td> 14170088 </td><td></td></tr><tr><td> Baixa temperatura de gelificação Agarose, tipo VII-A </td><td> Sigma-Aldrich </td><td> A0701 </td><td></td></tr><tr><td> Butil cola de cianoacrilato, Vetbond </td><td> 3M </td><td> 1469 </td><td></td></tr><tr><td> Arruelas de aço inoxidável, 4 mm de diâmetro interno </td><td> DIY loja </td><td></td><td></td></tr><tr><td> Rastreador de celular verde CMFDA </td><td> Invitrogen </td><td> C7025 </td><td></td></tr></tbody></table>

ex vivo imaging of t cells in murine lymph node slices with widefield and confocal microscopes

Ingênua células T tráfego continuamente para órgãos linfóides secundários, incluindo linfonodos periféricos, para detectar antígenos expressos rara. A migração de células T em linfonodos é um processo complexo que envolve fatores tanto celulares e químicas, incluindo quimiocinas. Recentemente, o uso de dois fótons de microscopia permitiu rastrear as células T nos linfonodos intactos e para obter alguma informação quantitativa sobre o seu comportamento e suas interações com outras células. Embora existam vantagens óbvias para uma no sistema vivo, esta abordagem requer uma instrumentação complexa e cara e oferece acesso limitado ao tecido. Para analisar o comportamento de células T dentro dos gânglios linfáticos murino, desenvolvemos um ensaio 1 fatia, originalmente criado pelos neurobiólogos e transposta recentemente para timo murino 2. Nesta técnica, fluorescente etiquetado células T são banhados em cima de uma fatia aguda preparado linfonodo. Neste vídeo-artigo, a localização e migração de células T no tecido são analisados ​​em tempo real com um campo amplo e um microscópio confocal. A técnica que complementa in vivo de dois fótons de microscopia oferece uma abordagem eficaz para células T imagem em seu ambiente natural e para elucidar os mecanismos subjacentes T migração celular.

Watch this Scientific Journal Video about Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes at JoVE.com

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Este protocolo descreve um método para células T imagem fluorescentes introduzido em fatias de linfonodos. A técnica permite análises em tempo real de migração de células T com o tradicional campo amplo de fluorescência ou microscópios confocal.

Ex vivo de imagens de células T em fatias Node murino linfáticos com Widefield e microscópios confocal

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

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Research

JoVE Journal

Immunology and Infection

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

16.7K visualizações.  Université Paris Descartes, CNRS (UMR 8104). Este protocolo descreve um método para células T imagem fluorescentes introduzido em fatias de linfonodos. A técnica permite análises em tempo real de migração de células T com o tradicional campo amplo de fluorescência ou microscópios confocal.

Vídeo: Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Transdução retroviral de receptor de células T no rato células T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

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 ...

Murine Superficial Lymph Node Surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

Cirurgia Nó murino superficial linfático

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Imagem intravital do Linfonodo Popliteal Rato

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Isolamento de Murino Linfonodo células estromais

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Um<em&gt; Ex vivo</em&gt; Modelo de um Ataque de T-Cell dirigiu-Oligodendrocyte em Agudos fatias de cérebro

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

Ex Vivo Infecção de Murino Epiderme com vírus herpes simplex tipo 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

Isolamento e<em&gt; Ex Vivo</em&gt; Cultura de Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; Células T, γδ um Extrathymic αβT de células progenitoras

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

Murino Linfócitos Rotulagem por<sup&gt; 64</sup&gt; Cu-Antibody Receptor Segmentação para<em&gt; In Vivo</em&gt; Tráfico celular por PET / CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...