Os autores gostaria de agradecer James Fosse pelo seu excelente trabalho em todos os Videografia e processamento de vídeo; Michael Marti pelo seu excelente trabalho na geração de imagens digitalizadas de gado; Lilia Walther por sua ajuda com a extração do RNA e Bioanalyzer é executado; Mitch Palmer e Carly Kanipe para sua revisão útil e feedback sobre imagens de linfonodo; e os cuidados com animais e pessoal veterinário no centro nacional de doença Animal para todos de seu trabalho árduo e assistência com a pecuária e a preparação para necropsias.

Os autores têm sem conflitos de interesse a divulgar. Toda pesquisa é financiada com fundos intramurais do departamento de agricultura dos EUA, Agricultural Research Service. Todas as referências a produtos específicos são fornecidas com a finalidade de reprodutibilidade experimental e não representam qualquer endosso destes produtos pelo governo federal.

A maioria dos estudos de transcriptomic e os protocolos associados concentrar em amostras humanas, rato, rato ou post-mortem. No entanto, investigações em animais e animais selvagens fornecem uma ampla gama de oportunidades para a caracterização da resposta imune à doença, tanto como aplicáveis à medicina veterinária e, no que diz respeito a doenças zoonóticas, a saúde pública. Este protocolo fornecido um resumo das considerações-chave para extração de RNA de alta integridade de tecidos de animais de gra...

A sequenciação do ARN da transcriptoma de linfonodos fornece a oportunidade para caracterizar a resposta imune dos animais para uma variedade de agentes patogénicos. Enquanto esta metodologia tem sido utilizada extensivamente em camundongos, análises tem recentemente expandido para maiores mamíferos1,2. Pecuária/grandes animais linfonodos podem ser usados para caracterizar respostas do hospedeiro a uma infecção, não só para a sua utilização em estudos de susceptibilidade genética ou vacina e para a identificação de alvos para o desenvolvimento de drogas, mas também como sistemas modelo para estudos em humanos em doenças zoonóticas. Por exemplo, no caso da brucelose (uma doença bacteriana zoonótica que impactos meio milhão de pessoas ao redor do mundo cada ano), apesar do aumento significativamente os custos, estudos em ovinos ou caprinos são mais relevantes para a infecção humana e vacina humana desenvolvimento de modelos animais de laboratório. Modelos de infecção mouse recapitular a infecção do sistema reticuloendotelial, mas não os sinais clínicos característicos3.
Em experimentos com animais grandes em comparação com estudos de animais de laboratório, o processo de tecido colheita necessariamente envolve um atraso maior entre a eutanásia e a coleção de tecido, que apresenta um potencial desafio para a preservação da RNA de alta qualidade. O RNA intacto é essencial para a geração de dados transcriptomic biologicamente relevantes. A geração do RNA de alta qualidade das amostras de tecido é particularmente crítico para estudos de grande patógeno animal realizado em instalações de contenção. Tais estudos são inerentemente mais difíceis de executar, como eles não só exigem aprovado instalações e pessoal altamente treinado, mas também carregam custos financeiros significativos, que, dependendo do trabalho, que podem variar de dezenas a centenas de milhares de dólares. Estes tipos de estudos envolvem também uma colaboração interdisciplinar e o conhecimento interdisciplinar para a sua conclusão, adicionando à sua complexidade. Portanto, formação sobre, desenvolvimento e adesão a um sistema simplificado para a coleta de amostra e preservação fornece benefícios significativos para a jusante estudos moleculares de tecidos de animais infectados.
A coleção de linfonodos maiores apresenta desafios adicionais para a coleta de tecido, em comparação com a amostragem semelhante de murino linfonodos. A preparação para a excisão de amostra exige um conhecimento básico da anatomia do nó de linfa, incluindo as estruturas internas relevantes. A estrutura de um nó de linfa é composta por lóbulos linfoides rodeados por seios cheios de linfa. Estas estruturas são colocadas dentro de uma cápsula fibrosa, resistente. 4 um lóbulo linfoide é "anatômica e funcional unidade básica do nó de linfa" e é composto de folículos, uma unidade profunda cortical e cordões medulares e seios4 (figura 1A). Linfócitos B e T são home aos folículos e profundo corticais unidades, respectivamente. Essas estruturas fornecem um andaime 3D e facilitam a interação entre os linfócitos e antígeno ou células apresentadoras.
Grosseiramente, folículos e unidades profundo corticais podem ser identificadas na superfície de corte como eles contêm uma malha reticular mais densa e aparecem mais escuros do que os seios, que são compreendidos por uma malha reticular mais delicada e aparecem mais leves (figura 1B). Por convenção, os patologistas referem-se às regiões dos gânglios linfáticos como o córtex superficial (folículos), o paracórtex (fundo corticais unidades) e a medula (cordões medulares e seios). Um exame adequado das três regiões tem sido considerado como a melhor prática em diretrizes de exame patológico de rotina para linfonodos5. Observe que há uma variação considerável a consistência, tamanho e cor dos gânglios linfáticos, mesmo dentro de um único animal. Como idade de animais, os gânglios linfáticos tendem a diminuir de tamanho e tornam-se mais firme do que aqueles de animais mais jovens, normalmente devido a um aumento em seu tecido conjuntivo e uma redução do linfoide normal estrutura6,7.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57195/57195fig1.jpg"> Figura 1. Anatomia do nó de linfa. (A), esta imagem mostra a anatomia do nó de linfa, representando estruturas chaves. (B) ainda esta imagem mostra um nó de linfa bovina cortado em secção transversal. Destacam-se as estruturas relevantes/camadas que são visíveis a olho nu. <a href="https://www.jove.com/files/ftp_upload/57195/57195fig1large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
Dependendo a questão experimental, linfonodos diferentes será de interesse para a recolha e análise. Linfonodos periféricos são aqueles localizados profundamente no tecido subcutâneo. Nos bovinos, linfonodos periféricos ou superficiais frequentemente utilizados na prática clínica e experimental incluem parótida, submandibular, retrofaríngeos, pré-escapulares, pré-femurais (precrural) e superficial inguinal (supramamários nas fêmeas, escrotal nos machos) ( A Figura 2). Na tabela 1, as propriedades de chaves linfáticos superficiais, conforme descrito no gado sistema8, são descritas. Abaixo, alguns planos de recolha de linfonodo potenciais para doenças infecciosas bacterianas, de gado são apresentados como ponto de partida para a investigação.
Brucella abortus/Brucella melitensis: padrão necropsies por b. abortus-infectados de gado e b. melitensis-cabras infectadas no centro nacional de doença Animal recuperar supramamários das, prescapular e tecido de linfonodo parotídeo , tanto para a moagem para a enumeração de bactérias e para a preparação de RNA para o perfil de expressão acolhimento do RNA. B. abortus regularmente podem ser recuperados em cada um destes gânglios linfáticos em bovinos experimentalmente infectados9. A presença de bactérias em cada um desses tipos de nó de linfa pode ser detectada em b. melitensis-infectados cabras até pós-infecção pelo menos nove meses, utilizando as metodologias baseadas em RNA de nossos estudos (Boggiatto et al, inédito). Salmonela SP.: O prescapular, subiliac (pré-femurais), e linfonodos mesentéricos foram úteis durante a criação de perfil de carcaças de gado para uma prevalência de Salmonella 10,11,12 e seria de interesse potencial para estudos de transcriptomic. Escherichia coli O157: H7: linfonodos mesentéricos (no intestino médio e intestino delgado distal locais) podem ser os sites de uma recuperação ocasional das bactérias em bezerros infectados (mas não em bovinos adultos infectados)13. Leptospirose (Leptospira SP.): uma crônica persistência das bactérias tem sido observada em linfonodos drenam a glândula mamária14. Mycobacterium bovis : Nos bovinos, as bactérias foram recuperada infecção pós-experimental de gânglios linfáticos do mediastino e traqueobrônquica de bezerros15. Além disso, o linfonodo RNA tem sido utilizado para examinar as respostas de grande animal hospedeiro de vírus, tais como a síndrome respiratória e reprodutiva suína vírus2. A Figura 2 mostra a localização de um subconjunto desses principais linfonodos do corpo de bovinos.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57195/57195fig2.jpg"> Figura 2: Cartoon retratando locais selecionados do nó de linfa no Bos taurus . Os linfonodos numerados são anotados. <a href="https://www.jove.com/files/ftp_upload/57195/57195fig2large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
Este documento e o vídeo associado, apresentamos um protocolo para o isolamento de grandes linfonodos animais para estudos de RNA, projetados para ser informativo para biólogos moleculares envolvidos em estudos de transcriptomic de infecções de animais grandes. Primeiro, nós fornecemos uma visão geral do procedimento de isolamento para os gânglios linfáticos, usando amostragem de tecidos bovinos e bison como exemplos. Emparelhado com esta demonstração, conforme exibido no vídeo, é um fluxo de trabalho para uma amostra de tecido pode ser reproduzido para isolamento de RNA. Em seguida, descrevemos considerações importantes para o processamento de um linfonodo infectado, com foco na segurança, consistência e qualidade do RNA.
A preparação do RNA do tecido com um reagente de isotiocianato de guanidina-fenol acidificado é baseada no método original de Chomczynski e Sacchi16,17, com uma purificação sobre colunas de rotação silicone-baseado na presença de agentes de caotrópicas baseados no trabalho de Vogelstein e Gillespie18original. Também examinamos o potencial para a recuperação do RNA para transcriptomics de gado gânglios linfáticos preservados por métodos alternativos. Finalmente, podemos explorar o impacto da variável tempo sobre a qualidade do RNA em necropsias de animais grandes, incluindo uma experiência representativa, retratando o efeito de um aumento no tempo entre a eutanásia e a amostragem no perfil de RNA recuperado de bison e linfonodos de bovinos. Este artigo será útil não apenas para biólogos moleculares, mas também ao veterinários pesquisadores iniciando estudos transcriptomic.

<table><tbody><tr><td>RNA preservation solution (we used RNALater for all experiments)</td><td>ThermoFisher</td><td>AM7020</td><td></td></tr><tr><td>1.5 ml or 2 ml polypropylene microcentrifuge tubes&amp;nbsp;</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Disposable scalpels</td><td>Daigger Scientific</td><td>EF7281</td><td></td></tr><tr><td>Tissue forceps, rat tooth</td><td>Fisher Scientific</td><td>12-460-117</td><td>Other tissue forceps available including curved tip, tapered edge, etc. , depends on user preference</td></tr><tr><td>3 mm punch biopsy needles</td><td>Fisher Scientific</td><td>NC9949469</td><td></td></tr><tr><td>Sharps container (small and transportable for necropsy)</td><td>Stericycle</td><td>8900SA</td><td>1 qt. size shown here</td></tr><tr><td>Cutting boards or disposable trays</td><td>Fisher Scientific</td><td>09-002-24A</td><td>Available in a variety of sizes, depends on user preference</td></tr><tr><td>Personal protective equipment</td><td></td><td></td><td>Varies with pathogen (gloves, respirator masks, goggles, etc.)</td></tr><tr><td>Phenol-based RNA extraction reagent (we used TRIzol Reagent for all experiments)</td><td>ThermoFisher</td><td>15596026</td><td></td></tr><tr><td>Silica column-based RNA extraction kit (we used the PureLink RNA Mini kit for all experiments)</td><td>ThermoFisher</td><td>12183018A</td><td>Designed for up to 100 mg tissue</td></tr><tr><td>100% Ethanol (200 proof for molecular biology)</td><td>Sigma-Aldrich</td><td>E7023</td><td></td></tr><tr><td>Tissue homogenizer with enclosed homogenization tubes (we used the gentleMACS dissociator for all experiments)</td><td>Miltenyi Biotec</td><td>130-093-235</td><td></td></tr><tr><td>Agarose (General, for gel electrophoresis)</td><td>Sigma-Aldrich</td><td>A9539</td><td></td></tr><tr><td>1X TBE</td><td>Fisher Scientific</td><td>BP24301</td><td>Can also make from scratch in the laboratory</td></tr><tr><td>Deionized formamide</td><td>EMD Millipore</td><td>S4117</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma-Aldrich</td><td>L3771</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma-Aldrich</td><td>114391</td><td></td></tr><tr><td>Xylene cyanol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>X4126</td><td></td></tr><tr><td>EDTA (Ethylenediaminetetraacetic acid)</td><td>Sigma-Aldrich</td><td>EDS</td><td></td></tr><tr><td>UV-Vis Spectrophotometer (we used the NanoDrop Spectrophotometer)</td><td>ThermoFisher</td><td>ND-2000</td><td></td></tr><tr><td>Device for quantitative RNA assessment (we used the Bioanalyzer, with associated components and protocols)</td><td>Agilent</td><td>G2939BA</td><td></td></tr><tr><td>FFPE RNA extraction kit (we used the RecoverAll Total Nucleic Acid Isolation Kit for Formalin Fixed, Paraffin Embedded Tissue)</td><td>ThermoFisher</td><td>AM1975</td><td></td></tr><tr><td>Plastic spreader (L-shaped spreader)</td><td>Fisher Scientific</td><td>14-665-231</td><td>Only needed for sterility testing for samples from infected animals</td></tr><tr><td>Necropsy knives</td><td>Livestock Concepts</td><td>WI-0009209</td><td></td></tr></tbody></table>

collection and processing of lymph nodes from large animals for rna analysis: preparing for lymph node transcriptomic studies of large animal species

Animais de grandes porte (gado e animais selvagens) servem como importantes reservatórios de agentes patogénicos zoonóticos, incluindo Brucella, Mycobacterium bovis, Salmonellae Escherichia colie são úteis para o estudo da patogênese e/ou propagação das bactérias em hospedeiros naturais. Com a função chave de linfonodos na resposta imune do hospedeiro, linfonodo tecidos servem como uma fonte potencial de RNA para análises de transcriptomic a jusante, a fim de avaliar as mudanças temporais na expressão gênica em células no decorrer de uma infecção. Este artigo apresenta uma visão geral do processo de coleção de nó de linfa, amostragem de tecido e a jusante do RNA processamento em gado, usando bovinos (Bos taurus) como um modelo, com exemplos adicionais provenientes do Bisão-americano (bison Bison ). O protocolo inclui informações sobre a localização, identificação e remoção dos gânglios linfáticos de diversos sites chaves no corpo. Além disso, uma metodologia de amostragem de biópsia é apresentada que permite a uma consistência de amostragem em vários animais. Várias considerações para preservação de amostra são discutidas, incluindo a geração de RNA apropriado para jusante metodologias como a sequenciação do ARN e RT-PCR. Devido os longos atrasos inerentes a estudos de curso de tempo grandes animais vs rato, apresentam-se resultados representativos de bison e tecidos de bovinos do nó de linfa para descrever o tempo de degradação neste tipo de tecido, no contexto de uma revisão da trabalho metodológico anterior na degradação de RNA em outros tecidos. Em geral, este protocolo será útil para ambos os pesquisadores veterinários começando transcriptome projetos em grandes amostras de animais e biólogos moleculares interessados em aprender técnicas para processamento de em vitro e em vivo amostragem de tecido.

O uso das considerações apresentadas neste artigo (passos 1-4 do protocolo) vai ajudar na recuperação do RNA de grandes amostras de animais que é adequada para uma análise a jusante em estudos de expressão de gene de anfitrião. A qualidade do RNA para aplicações a jusante é avaliada através de várias medidas padrão. Por espectrofotometria, a uma260/A280 ratio fornece uma medida da contaminação da proteína, e a uma260/A230 ratio fornece um outro meio de avaliaç...

Watch this Scientific Journal Video about Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species at JoVE.

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Coleta e processamento de linfonodos de animais de grandes porte para a análise do RNA: preparando-se para estudos de linfonodo Transcriptomic de espécies de animais grandes

Este protocolo fornece uma visão geral dos procedimentos para o isolamento do RNA para o perfil de transcriptomic do nó de linfa de tecidos de animais de grandes porte, incluindo etapas na identificação e excisão dos gânglios linfáticos do gado e animais selvagens, abordagens de amostragem para fornecer consistência em vários animais e considerações mais resultados representativos para a preservação de pós coleta e processamento para a análise do RNA.

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...

collection-processing-lymph-nodes-from-large-animals-for-rna-analysis

Research

JoVE Journal

Immunology and Infection

26.2K visualizações.  U.S. Department of Agriculture. Este protocolo fornece uma visão geral dos procedimentos para o isolamento do RNA para o perfil de transcriptomic do nó de linfa de tecidos de animais de grandes porte, incluindo etapas na identificação e excisão dos gânglios linfáticos do gado e animais selvagens, abordagens de amostragem para fornecer consistência em vários animais e considerações mais resultados representativos para a preservação de pós coleta e processamento para a análise do RNA.

Vídeo: Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

Não-cirúrgico instilação endotraqueal de camundongos com Análise de Pulmões e Drenagem Linfática Nodes Lung por citometria de fluxo

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

Imunologia e Infecção

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response 1. Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 2. Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37&deg;C.

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.

Dissecção e 2-Photon Imagens de linfonodos periféricos em ratos

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the ...

Biologia

Mouse Adipose Tissue Collection and Processing for RNA Analysis

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and Western Blot, since it mostly contains lipids. RNA isolation from adipose tissue samples is also challenging as extra steps are required to avoid these lipids. Here, we present a procedure to collect three anatomically different white adipose tissues from mice, to process these samples and perform RNA isolation. We further describe the synthesis of cDNA and gene expression experiments using real-time PCR. The hereby described protocol allows the reduction of contamination from the animal's hair and blood on fat pads as well as cross-contamination between different fat pads during tissue collection. It has also been optimized to ensure adequate quantity and quality of the RNA extracted. This protocol can be widely applied to any mouse model where adipose tissue samples are required for routine experiments such as real-time PCR but is not intended for isolation from primary adipocytes cell culture.

The purpose of this paper is to present a step-by-step procedure to collect different white adipose tissues from mice, process the fat samples and extract RNA.

Coleção de tecido adiposo de mouse e processamento para análise de RNA

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and ...

Bioquímica

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

Medição do Ca citosólico 2 + Em Isolated linfáticos contráteis

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

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 ...

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 ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

Identificação de codificação e Classes de RNA não-codificante expressa em suínos, sangue total

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Laparoscopic Technique for Serial Collection of Liver and Mesenteric Lymph Nodes in Macaques

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them immunologically unique. The GI tract and associated MLN are sites of early viral replication in human immunodeficiency virus (HIV) infection and the MLN are likely important reservoir sites that harbor latently-infected cells even after prolonged antiretroviral therapy (ART). The liver has been shown to play a significant role in immune responses to lentiviruses and appears to play a significant role in clearance of virus from circulation. Nonhuman primate (NHP) models for HIV and Acquired Immunodeficiency Syndrome (AIDS) closely mimic these aspects of HIV infection and serial longitudinal sampling of primary sites of viral replication and the associated immune responses in this model will help to elucidate critical events in infection, pathogenesis, and the impact of various intervention strategies on these events. Current published techniques to sample liver and MLN together involve major surgery and/or necropsy, which limits the ability to investigate these important sites in a serial fashion in the same animal. We have previously described a laparoscopic technique for collection of MLN. Here, we describe a minimally invasive laparoscopic technique for serial longitudinal sampling of liver and MLN through the same two port locations required for the collection of MLN. The use of the same two ports minimizes the impact to the animals as no additional incisions are required. This technique can be used with increased sampling frequency compared to major abdominal surgery and reduces the potential for surgical complications and associated local and systemic inflammatory responses that could complicate interpretation of results. This procedure has potential to facilitate studies involving NHP models while improving animal welfare.

Here, we describe a minimally invasive laparoscopic technique for serial sampling of liver and mesenteric lymph nodes (MLN) in macaques that allows for increased sampling frequency, and reduces the potential for surgical complications when compared to performing a laparotomy.

Técnica laparoscópica para a coleção de série do fígado e mesentérica linfonodos em macacos

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them ...

Medicina

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response against helminth infections, where it plays an important role in the expulsion of the parasite. In a murine model of Nippostrongylus brasiliensis infection, IL-9 is produced mainly by CD4+ T lymphocytes and innate lymphoid cells found in the lung, small intestine, and draining lymph nodes. Given the technical difficulties involved in the intracellular staining of IL-9, as well as the complexity of isolating hematopoietic cells from the small intestine upon infection, there is a pressing need for a comprehensive but straightforward protocol to analyze the expression of IL-9 in different lymphoid and non-lymphoid tissues in this model. The protocol described here outlines the kinetics of IL-9 produced by CD4+ T cells and innate lymphoid cells in the lung and small intestine, the main organs targeted by N. brasiliensis, as well as in the mediastinal and mesenteric lymph nodes, throughout the infection. In addition, it details the number of larvae needed for infection, depending on the cell type and organ of interest. This protocol aims to assist in the standardization of assays to save time and resources by offering the opportunity to focus on the specific cells, organs, and disease stages of interest in the N. brasiliensis infection model.

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9-expressing T and ILC2 cells are induced during N. brasiliensis infection, yet their characterization has been largely overlooked in the infected intestine due to their low frequency and differential kinetics. This protocol describes the isolation of these cells from different target organs and confirmation of their identity via flow cytometry at different infection stages.

Uma estratégia para o estudo de células linfoides produtoras de IL-9 no modelo de infecção por Nippostrongylus brasiliensis

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response ...

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important phenomenon in the gut immunity. The stroma of secondary lymphoid organs, such as Peyer&#8217;s patches (PPs) or mesenteric lymph nodes, are where na&#239;ve lymphocytes sense antigens. Na&#239;ve lymphocytes circulate through the bloodstream to reach high endothelial venules, the portal of entry into PPs. Some immunomodulators are estimated to influence lymphocyte migration, but the precise evaluation of microcirculation dynamics is very difficult, and establishing a method to observe lymphocyte migration in vivo can contribute to the clarification of the precise mechanisms. We refined the method of collecting lymphocytes from the lymph duct and observing the detailed dynamics of gut-tropic lymphocytes in rat PPs. We chose confocal laser scanning microscopy to observe rat PPs in vivo and recorded it using time-lapse photography. We can now obtain clear images that can contribute to the analysis of lymphocyte dynamics.

Here, we describe a precise method for collecting thoracic duct lymphocytes and observing the migration of gut-tropic lymphocytes in rat Peyer&#8217;s patches for 3 hours using time-lapse photography. This technique can clarify how the dynamics of lymphocytes are affected under inflammatory conditions.

Observação microscópica da Dinâmica do Linfócito em Patches de Rat Peyer

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important ...