1. Antes do processo, preparar e recolher os itens a seguir <ol><li> Por favor, consulte a imagem da Figura 1a) e 1b) para a construção de uma plataforma de madeira para conter o mouse durante o procedimento. </li><li> Mix digestão para os gânglios linfáticos - HBSS + 1.25mg/ml colagenase TypeIV ou 2,5 mg / ml de colagenase D </li><li> Mix digestão para os pulmões - HBSS + 1 colagenase mg / ml </li><li> Diluir esferas de látex (1:20) em PBS para injeção intraperitoneal para visualizar a LDLNs </li></ol> Nota: LDLNs são pequenas e difíceis de encontrar. Para saber como encontrá-los, injetar 200μl das 01:20 diluída vermelho esferas de látex fluorescentes ip para o mouse. O peritônio é drenada para essas LDLNs 8. 24 hpi, expor a cavidade torácica do mouse, como descrito acima. O LDLNs aparecerá rosa das contas que drenada da cavidade peritoneal. <ol start="5"><li> Glóbulos vermelhos (RBC) Tampão de Lise - 0,15 M NH 4 Cl, 10mM KHCO 3, 0,1 mM Na 2 EDTA em 1L dH 2 0. Ajustar o pH a 7,2-7,4 e armazenar em temperatura ambiente. </li><li> Coador de células 70μm ou 100μm </li><li> Solução estoque de EDTA 0,5 M, pH = 8,0 </li><li> HBSS sem CaCl 2 e MgCl 2 </li><li> Tesouras e pinças de ponta e </li><li> Laringoscópio </li><li> Agulha de gavage dobrado (calibre 22) em um ângulo (Figura 2) </li><li> Isopor placa e pinos para espalhar o rato </li><li> Equilíbrio pan topo </li><li> Solução de anilina azul - 10mg/ml em H 2 O </li></ol> Nota: Nós estaremos usando esta solução para a demonstração de que a técnica de injecção. 2. Preparação e administração de anestésico do mouse <ol><li> Prepare uma mistura de xilazina e quetamina em PBS estéril, para que as concentrações finais são 2mg/ml e 10mg/ml respectivamente. </li><li> Pesar o mouse em uma balança de prato superior. </li><li> Calcular a dose necessária para cada rato. Dose de Xilazina (2-4 mg / kg) e ketamina (70-80 mg / kg) de peso corporal usando volume apropriado da mistura preparada e injetar intaperitoneally. Cada cepa do mouse pode exigir otimização de anestésico. Camundongos que receberam muito anestésico pode ter uma taxa de respiração reduzida que poderia ser fatal depois de inóculo é instilada nos pulmões. </li><li> Depois de 3-5 minutos, observe o mouse para os efeitos do anestésico. Verifique os seguintes sinais para confirmar que o mouse é totalmente anestesiado: <ol><li> A taxa de respiração deve desacelerar. </li><li> O rato não deve esticar seu braço quando pegou pelo pescoço. </li><li> Não deve haver qualquer resposta quando a membros posteriores são estimulados. Se estes critérios não forem atendidos, espere alguns minutos e verifique novamente antes de prosseguir para a próxima etapa. </li></ol></li></ol> 3. Injeção intratraqueal <ol><li> Preparar o inóculo em salina 50μl. Encher uma seringa de 1ml com uma agulha estéril dobrada gavage com o inóculo. A seringa deve ser carregado com uma bolsa de ar 100μl por trás do inóculo para garantir que todo o líquido é instilado no pulmão. </li><li> Posicione o mouse sobre a plataforma de madeira em ângulo pendurado pelas incisivos no fio e gentilmente contê-lo no lugar com um pedaço de fita. </li><li> Ligue o laringoscópio com a mão esquerda (se você for destro) e pegar um par de fórceps blunt terminou. Use a ponta do laringoscópio e as pinças para gentilmente forçar a abertura da boca. </li><li> Com a pinça, puxar a língua para fora e segure-o para o lado. Guia da lâmina do laringoscópio para a parte traseira da boca. Mantenha o laringoscópio pressionado muito suavemente em um ângulo de 90 ° até ver a abertura da traquéia. Segure o laringoscópio no lugar. Cuidados devem ser tomados quando puxar a língua da boca e isso deve ser feito com cuidado para não danifica o tecido ocorre. </li><li> Com a outra mão, pegue a seringa de 1ml contendo o inóculo, equipado com a agulha torta gavage com as mãos direita e inserir a agulha para dentro da traquéia até a curva da agulha é por os incisivos da frente. Empurre o êmbolo uniformemente para entregar o inóculo. O inóculo não deve bolha. Retire a agulha da traquéia, logo que possível. Camundongos vai sufocar e morrer se a traquéia é bloqueada por muito tempo. Segure o mouse na vertical por alguns segundos para permitir inóculo para ser inalado para os pulmões. </li><li> Leve o mouse para fora da plataforma e colocá-lo perto de uma lâmpada de aquecimento. Não deixe os ratos sob o calor direta por longos períodos de tempo. O mouse deve ser totalmente desperto dentro de 30 min após o procedimento (tempo de recuperação pode variar de acordo com cepas). Camundongos devem ser observados com diligência durante este tempo e não devem ser autorizados a ficar muito quente ou muito frio, isso também poderia colocar os ratos em angústia respiratória levando à morte. </li></ol> 4. Colheita e processamento de pulmão linfonodos drenantes e pulmões para a citometria de fluxo<ol><li> Sacrifício camundongos usando o método desejado de acordo com as diretrizes da eutanásia AVMA. Isso pode variar, dependendo dos resultados da colheita individual. Administração de um letal de pentobarbital sódico não é um exemplo de um procedimento de eutanásia apropriado. Não temos visto efeitos negativos pulmonar de eutanásia por CO 2. </li><li> Pin dos braços e pernas em uma placa de dissecção. Faça uma incisão ao longo do meio do lado ventral com uma tesoura. Puxe a pele e expor os músculos da parede torácica. </li><li> Fazer incisões através dos músculos da parede torácica para expor os órgãos abdominais. </li><li> Segure a ponta do esterno com a pinça e punção do diafragma. Corte o diafragma ao longo dos lados da caixa torácica. </li><li> Com a tesoura paralela à superfície da placa de dissecação, corte através da cavidade torácica ao longo da linha dorso-ventral em ambos os lados. </li><li> Levante ligeiramente a caixa torácica para cima. No lado direito do coração, logo abaixo do timo, olhar para dois linfonodos. Um nó de linfa ligeiramente maior terceiro está localizado por baixo um pequeno vaso sanguíneo arterial que corre perpendicular à traquéia. Colheita gânglios linfáticos em 1 ml de linfa mix digestão nó. </li><li> Após a colheita os gânglios linfáticos, segure o coração com uma pinça e pegar os pulmões com o outro. Pulmões colheita em 3 ml de PBS gelado em uma placa de 6 poços. </li><li> Separam os lobos dos pulmões, aspirado PBS e pique os lobos em pedaços pequenos usando uma pinça e tesouras. Adicionar 3 ml da mistura de digestão para os pulmões. Mantenha a placa de 6 poços em gelo durante esta etapa. </li><li> Incubar os linfonodos por 25-30 min e pulmões por 30 min a 37 ° C. Adicionar EDTA para uma concentração final de 10mM de cada uma das misturas digestão para parar a reação e colocar no gelo. </li><li> Usando uma seringa de 3ml, passar pedaços de pulmões através de uma agulha 18GA 3 vezes. Mash as peças dos pulmões e os linfonodos através de filtros de 70 100μm célula usando o back-end de um êmbolo da seringa de 1ml. Lavar os filtros de células com 10 ml de HBSS sem Ca 2 + e Mg 2 +. Centrifugar a 500 xg por 5 min. </li><li> Ressuspender o sedimento em 5 ml de tampão de lise RBC e incubar por 3 min à temperatura ambiente. Adicionar 10 ml de HBSS sem Ca 2 + e Mg 2 + para saciar a lise. Centrifugar a 500 xg por 5 min. Abrir o pulmão e suspensões de células LDLN único para 10ml e 5ml respectivamente. Contagem de células vivas usando um hemocitômetro por exclusão de tripan azul. Células estão agora prontos para mancha de citometria de fluxo. Um lobo do pulmão pode ser suficiente para citometria de fluxo. Outros lobos pode ser usado para outras questões experimentais na mesmo mouse, como homogenatos de citocinas ou tecido para exame histológico. </li></ol> 5. Resultados representativos: PMA e macrófagos alveolares nos pulmões adquirir antígeno dos espaços alveolares dos pulmões imediatamente a seguir que a injeção. A Figura 3 mostra percentuais de CD11c + (expressa em países menos desenvolvidos e nos macrófagos alveolares), as células que são positivas para os esporos de Bacillus anthracis fluorescente etiquetado, pós 30 min que a injeção. Neste momento, ~ 15,9% de CD45 + CD11c + células em suspensão de células de pulmão único abrigou esporos de Bacillus anthracis (Figura 3, painéis inferiores). Para uma melhor caracterização de células CD11c + nos pulmões, referem-se a Kim e Braciale 4. Fluorescente etiquetado contas são rotineiramente usados ​​para avaliar o tráfico de antígenos particulados para LDLNs 9. Para avaliar este transporte dos pulmões para LDLNs, miçangas fluorescentes de látex foram injetados com LPS e LDLNs foram analisados ​​em 24 hpi. Cerca de 4% de células CD11c oi na LDLNs abrigou partículas fluorescentes em 24hpi (Figura 4). Estas experiências mostram representante que este protocolo pode ser eficientemente usado para rastrear células que adquirir antígenos particulados inalado nos pulmões e, posteriormente, o tráfego los para o LDLNs. <img src="/files/ftp_upload/2702/2702fig1.jpg" alt="Figura 1"/> Figura 1. Plataforma de madeira para imobilizar ratos durante a mesma injeção. Um bloco de madeira foi cortada em um ângulo e barras de metal foram instalados, front view figura 1a) e laterais figura 1b vista). Um pequeno pedaço de fio foi enrolado ao redor da barra de metal para ancorar os incisivos frente do mouse. Um pedaço de fita foi anexada à plataforma para amarrar ao redor do mouse para mantê-lo no lugar durante o procedimento. <img src="/files/ftp_upload/2702/2702fig2.jpg" alt="Figura 2"/> Figura 2. Gavagem agulha de injeção para ele. Uma agulha gavage (direita) foi dobrada em um ângulo de aproximadamente 135 ° (esquerda) para que a injeção. <img src="/files/ftp_upload/2702/2702fig3.jpg" alt="Figura 3"/> Figura 3. Porcentagem de todas as células CD11c + nos pulmões que porto se expressando GFP injetado Bacillus anthracis (Sterne-p6GFP), o desafio pós 5hrs. Camundongos foram AJ desafio com 1x10 8 esporos GFP Bacillus anthracis, e os pulmões foram analisados ​​em 5 horas após a infecção. Os gráficos de pontos mostram porcentagem de CD11c + células do pulmão que são positivos para o Bacillus anthracis. <img src="/files/ftp_upload/2702/2702fig4.jpg" alt="Figura 4"/> Figura 4. Detecção de partículas fluorescentes (amarelo / verde) no pulmão linfonodos drenantes 24 horas após a i. Injeção t. AJ ratos receberam ou PBS (painel esquerdo) ou 5x10 9 Fluoresbrite Microesferas YG (0.5μm) com 10μg de LPS (painel direito) que foram analisados ​​e LDLNs 24hpi. Parcelas representativas dot mostram porcentagem de células CD11c oi na LDLNs que contas porto fluorescentes conspiraram contra um canal vazio.

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Não há conflitos de interesse declarados.

Nós utilizamos esse protocolo para estudo do tráfico de esporos de Bacillus anthracis dos pulmões para o LDLNs. Para aplicações semelhantes, o número de partículas entregues para os pulmões devem ser cuidadosamente escolhidos para que o material injetado pode ser detectado no LDLNs por citometria de fluxo. Temos também utilizado com sucesso este método para a transferência adotiva de células e rotulagem das populações específicas de células do pulmão através de anticorpos fluorescente etiqueta...

<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> Stariner celular 70μm </td><td> BD </td><td></td><td></td></tr><tr><td> Anilina Azul </td><td> Pescador </td><td> A967-25 </td><td></td></tr><tr><td> Agulha de Alimentação Animal </td><td> Popper and Sons Inc </td><td> 7920 </td><td></td></tr><tr><td> Colagenase </td><td> Sigma </td><td> C2139 </td><td></td></tr><tr><td> Colagenase TypeIV </td><td> Worthington </td><td></td><td></td></tr><tr><td> Colagenase D </td><td> Roche </td><td> 11088974103 </td><td></td></tr><tr><td> DPBS </td><td> Invitrogen </td><td> 14190 </td><td></td></tr><tr><td> Fluoresbrite Microesferas YG (0.5μm) </td><td> Polysciences, Inc </td><td> 17152 </td><td></td></tr><tr><td> HBSS sem cloreto de cálcio e cloreto de magnésio </td><td> Invitrogen </td><td> 14170 </td><td></td></tr><tr><td> Ketamina HCl (100mg/ml) </td><td> Hospira Inc </td><td></td><td></td></tr><tr><td> Lâmina de laringoscópio </td><td> PennCentury, Inc </td><td> Para o modelo LS-1 </td><td> Referem-se a <a href="http://www.penncentury.com">www.penncentury.com</a> </td></tr><tr><td> Laringoscópio de Fibra Óptica leve </td><td> WelchAllyn </td><td> 80814 </td><td></td></tr><tr><td> Red Beads Fluorescente (0.5μm) </td><td> Invitrogen </td><td> F8812 </td><td> Para injeção ip </td></tr><tr><td> Xilazina (100mg/ml) </td><td> Lloyd Laboratories </td><td></td><td></td></tr></tbody></table>

non-surgical intratracheal instillation of mice with analysis of lungs and lung draining lymph nodes by flow cytometry

Células fagocíticas, tais como macrófagos alveolares e células dendríticas pulmonares (PMD) continuamente amostra antígenos dos espaços alveolares nos pulmões. PMD, em particular, são conhecidos a migrar para o pulmão linfonodos de drenagem (LDLNs) onde apresentam antígenos inalados para células T iniciar uma resposta imune apropriada para uma variedade de imunógenos 1,2. Para modelar interações entre os pulmões e antígenos no ar em camundongos, os antígenos podem ser administradas por via intranasal 1,3,4, intratraqueal 5 ou 6 como aerossóis. Entrega por cada rota envolve distintas habilidades técnicas e limitações que precisam ser considerados antes de projetar um experimento. Por exemplo, a exposição intranasal e aerossol oferece antígenos para ambos os pulmões e do trato respiratório superior. Daí antígenos pode acessar o tecido linfóide associado nasal (NALT) 7, potencialmente complica a interpretação dos resultados. Além disso, engolir espirros, ea taxa de respiração do mouse também pode levar a inconsistências nas doses administradas. Embora o envolvimento do trato respiratório superior pode ser preferível para alguns estudos, pode complicar experimentos com foco em eventos especificamente iniciada nos pulmões. Neste cenário, a rota (it) intratraqueal é preferível, uma vez que fornece materiais de teste diretamente para os pulmões e ignora o NALT. Muitos protocolos que envolvem injeção de intubação ou cego da traquéia através da cavidade oral ou exposição cirúrgica da traquéia para acessar os pulmões. Relata-se um simples, consistente, o método não-cirúrgico para que a instilação. A abertura da traquéia é visualizado através de um laringoscópio e uma agulha torta sonda é inserido diretamente na traquéia para entregar o inóculo. Descrevemos também os procedimentos de colheita e processamento de LDLNs e pulmões para a análise do tráfico de antígeno por citometria de fluxo.

Watch this Scientific Journal Video about Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry at JoVE.com

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

Nós ilustramos não-cirúrgico de entrega de materiais de teste para os pulmões de ratos anestesiados através da traquéia. Este método permite a exposição de pulmão a patógenos virais e bacterianas, citocinas, anticorpos, miçangas, químicos ou corantes. Iremos descrever colheita e processamento dos pulmões e dos gânglios linfáticos de drenagem de pulmão (LDLNs) para citometria de fluxo.

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, ...

non-surgical-intratracheal-instillation-mice-with-analysis-lungs-lung

Research

JoVE Journal

Immunology and Infection

57.0K visualizações.  University of Colorado School of Medicine. Nós ilustramos não-cirúrgico de entrega de materiais de teste para os pulmões de ratos anestesiados através da traquéia. Este método permite a exposição de pulmão a patógenos virais e bacterianas, citocinas, anticorpos, miçangas, químicos ou corantes. Iremos descrever colheita e processamento dos pulmões e dos gânglios linfáticos de drenagem de pulmão (LDLNs) para citometria de fluxo.

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

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

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

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

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

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

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

Imunologia e Infecção

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Detecção de nanopartículas fluorescentes Interações com Primário imunitário subpopulações de células por citometria de fluxo

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

Um ensaio baseado em CFSE para estudar a migração de Murino pele as células dendríticas em drenagem gânglios linfáticos durante a infecção com<em&gt; Mycobacterium bovis</em&gt; Bacilo de Calmette-Guérin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension6. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.

This article provides detailed methodology to identify and quantify functional T lymphocyte subsets present within murine kidney, aorta and lymph nodes by intracellular staining and flow cytometry. The model of angiotensin II induced hypertension was chosen to explain, step-by-step, the procedures and fundamental principles of flow cytometry and intracellular staining.

Intracelular Coloração e Citometria de Fluxo para identificar linfócitos subconjuntos dentro Murino Aorta, rim e linfonodos em um modelo de hipertensão

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our ...

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

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

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

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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolamento e cultura In Vitro de macrófagos alveolares murino e humanos

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Caracterização de subconjuntos de monócitos humanos por sangue total Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

Determinação de subconjuntos de regulação de células T no timo murino, pancreático drenagem do linfonodo e baço usando citometria de fluxo

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

Preparação de medula óssea inteira para análise de citometria de massa de células de linhagem de neutrófilos

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

A Model of Self-limited Acute Lung Injury by Unilateral Intra-bronchial Acid Instillation

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic findings similar to human acute respiratory distress syndrome (ARDS). The resulting alveolar edema, alveolar-capillary barrier damage, and leukocyte infiltration predominantly affect the left lung, preserving the right lung as an uninjured control and allowing animals to survive. This model of self-limited acute lung injury enables investigation of tissue resolution mechanisms, such as macrophage efferocytosis of apoptotic neutrophils and restitution of alveolar-capillary barrier integrity. This model has helped identify important roles for resolution agonists, including specialized pro-resolving mediators (SPMs), providing a foundation for the development of new therapeutic approaches for patients with ARDS.

Selective intra-bronchial acid instillation to the left lung in mice results in unilateral and self-limited acute lung injury that models human acute respiratory distress syndrome (ARDS) induced by gastric acid aspiration.

Um modelo de lesão pulmonar aguda autolimitada por instilação unilateral de ácido intra-brônquico

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic ...