The authors thank Sanjiv Luther and colleagues for helpful discussions in establishing the current lymph node digestion protocol. This work was supported by SNF grants PPOOA-_119204 and PPOOP3_144918 to S.W.R.

Nesta publicação de vídeo e protocolo, todos os procedimentos com animais foram realizados de acordo com o protocolo de animais aprovados pela Autoridade Cantonal Basel-Stadt, Suíça. 1. Linfonodos Preparação e Digestão <ol><li> Pré-aquecimento de água num copo a 37 ° C num agitador magnético com placa de aquecimento. </li><li> Prepare médio básico como se segue: DMEM (sem piruvato) suplementado com 2% de FCS, 1,2 mM CaCl2 e Pen / Strep (100 unidades de penicilina, 100 ug de estreptomicina). </li><li> Esterilizar todos os instrumentos de dissecção antes do uso. </li><li> Eutanásia camundongos linfonodo doadores por asfixia com CO2 e assepticamente dissecar os gânglios linfáticos. Não dissecar a gordura ao redor. NOTA: Este protocolo é otimizado para o nó periférico de drenagem linfática da pele (inguinal, braquial, axilar). </li><li> Coloque gânglios linfáticos em uma placa de Petri esterilizada, contendo 2 ml de meio de base de gelo frio. </li><li> Interromper o linfonodo capsule usando duas agulhas 25 G fixos em 1 ml seringa. </li><li> Transferir o tecido de linfonodos interrompido num tubo de polipropileno de 5 ml de fundo redondo contendo 750 ul meio básico complementado com 1 mg / ml de colagenase IV, e 40 ug / ml de ADNase I. </li><li> Adicionar um agitador magnético estéril em cada tubo. </li><li> Colocar o tubo no recipiente com 37 ° C pré-aquecido de água e agita-se os tubos a uma velocidade lenta (1 volta / seg) durante 30 min. </li><li> Remover o tubo do agitador magnético com placa de aquecimento e deixar fragmentos de linfonodos resolver. </li><li> Remova cuidadosamente o sobrenadante enriquecido em &quot;células não-estromal&quot;. NOTA: Se a análise de T, B, células dendríticas e CD45 - gp38 - CD31 - está prevista, salvar a fração &quot;células não-estromais&quot;. </li><li> Lavar o tecido restante linfonodo uma vez com 750 ul meio básico. NOTA: Esta etapa é necessária somente se a trabalhar com camundongos imunocompetentes. </li><li> Vamos fragmentos de linfonodos resolver. </li><li> Remover ofracção flutuante de células não-estromal. </li><li> Adicionar aos fragmentos de nódulos linfáticos meio básico de 750 ul suplementadas com 3,5 mg / ml de colagenase D e 40 ug / ml de ADNase I. </li><li> Colocar o tubo de volta na taça que contém a 37 ° C pré-aquecido a água. </li><li> Digerir o tecido do linfonodo para 5 min, enquanto lentamente mexendo. </li><li> Linfáticos fragmentos de tecido nó desagregados por pipetagem e misturando 700 mL para 10 ciclos em máxima velocidade usando uma pipeta multicanal automatizada. NOTA: Isso atrapalha linfonodo para melhorar a digestão. </li><li> Colocar o tubo de volta no recipiente com água a 37 ° C pré-aquecido. </li><li> Fragmentos de tecidos de linfonodo Digest para mais 10 min, enquanto lentamente agitação. </li><li> Desagregar fragmentos de tecidos de nódulos linfáticos por pipetagem e mistura por 99 ciclos a velocidade máxima utilizando uma pipeta multicanal automatizada. </li><li> Adicionar 7,5 mL de 0,5 M de EDTA para garantir a manutenção da suspensão de célula única. </li><li> Fragmentos de tecidos de linfonodo desagregados por tubotting e mistura por 99 ciclos a velocidade máxima utilizando uma pipeta multicanal automatizada. </li><li> Adicionar meio básico de 750 ul e as células passam através de uma malha de nylon de 70 ^ m. </li><li> Centrifugar a suspensão de células de 5 min a 1500 xg, 4 ° C. </li><li> As células do estroma pode ser agora usado para posterior análise. </li></ol> 2 Coloração de Linfonodo células estromais <ol><li> Use uma combinação de anti-CD45, anti-podoplanina (gp38), os anticorpos anti-CD31 para reconhecer com sucesso células TRC, LEC, BEC e DN. </li><li> Incubar o tecido linfático nó digerido com vivo inoperante rastreador /, anti-CD45-FITC (1: 200), anti-gp38-PE (1: 200), anti-CD31-APC (1: 200) em 100 uL de HBSS contendo 2 % FCS para, pelo menos, 20 min, 4 ° C, no escuro. </li><li> Lave as células adicionando 500 l de HBSS contendo 2% de FCS </li><li> Centrifugar a suspensão de células a 3 minutos a 1500 xg, 4 ° C. </li><li> Re-suspender em 100 uL de HBSS contendo 2% de FCS. </li><li> Executar as células marcadas num citómetro de fluxo equed, com as seguintes óptica: fonte de excitação com até três lasers: azul (488 nm, arrefecido a ar, de 20 mW de estado sólido), vermelho (633 nm, 17 mW HeNe), e violeta (405 nm, 30 mW de estado sólido) . </li><li> Portão de CD45 - células para excluir as células hematopoiéticas. </li><li> Singlets Portão (FSC-W) e de células vivas (VIVO / rastreador DEAD) para excluir doublets e células mortas. </li><li> Plot gp38 contra CD31 para visualizar TRC (gp38 + CD31 -), LEC (gp38 + CD31 +), BEC (gp38 - CD31 +) células (ver Figura 1) e DN (gp38 - - CD31). </li></ol>

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The authors declare they have no competing financial interests.

O estudo das células do estroma dos nódulos linfáticos recentemente tornou-se um foco de investigação devido ao desenvolvimento de dois protocolos de digestão publicados 6,13. Ambos os protocolos são adequados para obter único nó de linfa de células estromais, mas diferem na utilização de enzimas de digestão e tempo de digestão. Uma vez que as células estromais e seus marcadores de superfície são sensíveis à digestão enzimática e tensão mecânica, um protocolo optimizado é necessária. ...

Os linfonodos são compartimentos especializados onde as respostas imunes adaptativas contra-antígenos próprios estrangeiros e são iniciadas e coordenadas. O processo aqui apresentado descreve uma digestão enzimática curto combinado com pipetagem automatizada mecânica para obter linfonodo suspensão de células individuais e obter acesso às células viáveis ​​do estroma de nódulos linfáticos, que mantêm a expressão de várias moléculas da superfície. Formar células do estroma linfonodo cadafalso do linfonodo e cumprir três funções principais: primeiro eles filtram líquidos do corpo para provar antígenos, patógenos e sua patógeno padrão molecular associado (PAMPs), bem como as citocinas e perigo associado padrão molecular (amortece) presente no corpo. Em segundo lugar, eles atraem e instruir as células apresentadoras de antígenos (APC) e linfócitos para interagir e iniciar respostas imunes adaptativas; e em terceiro lugar, que proporcionam um ambiente estrutural para a homeostase e diferenciação dos linfócitos 1-3. Durante a inflamação células do linfonodo estromais produzem fatores de crescimento, citocinas e quimiocinas, adaptar-se, assim, o inchaço organizar a interação entre células dendríticas (DCs), T e células B. A orquestração das respostas imunitárias só é possível devido a arquitectura estrutural complexo formado por diferentes populações de células do estroma. As células do estroma de nódulos linfáticos são células CD45 negativas e pode ser distinguida da expressão de CD31 ou gp38 em células fibroblásticas e endoteliais 1 -6. GP38 + CD31 - define as células da zona T reticulares (TRC, também conhecidos como FRC: células fibroblásticas reticulares), gp38 + CD31 + define células endoteliais linfáticas (LEC), gp38 - CD31 + define as células endoteliais do sangue (BEC). Além disso, a caracterização das subpopulações revelou a existência de outras células do estroma de nódulos linfáticos. De facto, uma pequena população de células-pericyte como foi caracterizado nagp38 - CD31 - população 7. Portanto, a adaptação do procedimento de isolamento é vantajosa para a identificação e caracterização das propriedades funcionais diferentes de células do estroma de nódulos linfáticos. Antes do desenvolvimento de linfonodo células estromais digestão protocolos que o estudo de células estromais de linfonodos foi limitado a observações in situ usando secção de tecido e microscopia. No entanto, estudos estruturais e funcionais apresentaram características importantes de células estromais de linfonodos. As células do estroma de nódulos linfáticos são associados com podoplanina, colagénio e proteínas (ECM) de matriz extracelular de modo a formar uma estrutura tridimensional complexa 3 chamado sistema de conduta, a qual transporta as proteínas linfáticas e massa molecular associada menor do seio subcapsular do nó de linfa para o alto endotélio vênulas na zona de células T 8. DCs estão em contato com células do estroma e pode ser observado saliente no con tubularestrutura duit a amostra de fluido e detectar antigénios 8. A interação das células do estroma dos nódulos linfáticos (TRCs e LECs) com DCs é mediada pela liberação e apresentação das quimiocinas CCL21 e CCL19 9,10. CCL19 e CCL21 são reconhecidos pelo receptor CCR7 facilitar DCs e as células T migrem para a zona das células T linfonodo 4,11. Apesar de usar quimiocinas semelhantes, as células T e DCs têm diferentes rotas de migração para os linfonodos 12. Mais tarde, utilizando a digestão enzimática do nódulo linfático e isolamento de células do estroma de nódulos linfáticos puros, estudos funcionais foram realizados sobre o papel dos diferentes células do linfonodo estroma e a sua capacidade para interagir com DC e células T / B 6,13. Em primeiro lugar, a interferência entre as células produtoras de IFN-T efectoras e células estromais γ nódulos linfáticos induz a produção do óxido nítrico metabolito mostrado para amortecer as respostas das células T e a proliferação dos órgãos linfóides secundários 14-16. Em segundo lugar, n linfaAs células do estroma ode ter sido relatado para suportar a diferenciação de subconjuntos de DC de regulação através da produção de IL-10, 17, e para modular a homeostase da célula T ingénuos através da produção de IL-7 6,18. Em terceiro lugar, a expressão de TLR em células do estroma de nódulos linfáticos sugerem que as células do estroma são susceptíveis ao sinal obtido a partir de uma infecção ou de auto-moléculas libertado durante a lesão de tecidos. De facto, o tratamento de células do estroma de nódulos linfáticos com o ligando de TLR3 de poli (I: C) induz uma regulação positiva modesta de grande expressão de histocompatibilidade classe I e complexa regulação positiva da molécula co-inibidor PD-L1, mas não das moléculas de co-estimulação, resultando em mudanças dramáticas no tecido periférico antígenos expressão 19. Diversos grupos têm apontado células do estroma dos nódulos linfáticos expressar antígenos de tecidos periféricos e induzir tolerância das células T auto-reactivas 19,21-27. Portanto, a compreensão das interacções entre células do estroma dos nódulos linfáticos e outro re migratório ecélulas do linfonodo sident vai ajudar a encontrar novas moléculas alvo para permitir a ativação ou supressão de respostas imunes durante a inflamação. Portanto, é necessária a implementação da separação enzimática publicado do linfonodo. Protocolos previamente publicados usar diferentes combinações de digestão enzimática à base de colagenase com baixo esforço mecânico 6,19,20. No entanto, longas incubações com enzimas da digestão ou a combinação de diferentes tipos de enzima de digestão pode prejudicar várias moléculas de superfície necessárias para analisar o status de ativação e identificar novas células estromais de linfonodos. Dependendo do tipo de análise de células do estroma, o protocolo de ligação ou Fletcher protocolo pode ser mais adequado. No processo descrito, de uma digestão enzimática ligeiramente mais curto é combinado com a desagregação mecânica automatizado para minimizar a degradação da superfície marcador linfáticos viável células estromais nó. Este procedimento permite que o isolamento altamente reprodutível edistinção de nódulos linfáticos de populações de células do estroma com uma baixa variabilidade e de mais do que 95% de viabilidade. As células do estroma de nódulos linfáticos isolados de fresco pode ser usado directamente para a expressão do marcador de superfície, a análise de proteínas, e estudos de transcrição, bem como o estabelecimento de linhas de células estromais de realizar os ensaios funcionais in vitro.

<table><tbody><tr><td>DMEM</td><td>LifeTechnologies-Gibco</td><td>41965-039</td><td></td></tr><tr><td>FCS</td><td></td><td></td><td>Final concentration 2%</td></tr><tr><td>CaCl2</td><td>Sigma</td><td>499609</td><td>Final concentration 1.2 mM</td></tr><tr><td>Collagenase IV</td><td>Worthington Biochemical Corporation</td><td></td><td>&gt;160 units per mg dry weight, use at final concentration of 1 mg/ml</td></tr><tr><td>Collagenase D</td><td>Roche</td><td>11088882001</td><td>use at 3.5 mg/ml</td></tr><tr><td>DNAse I</td><td>Roche&amp;nbsp;</td><td>11284932001</td><td>use at 40 &amp;micro;g/ml</td></tr><tr><td>Stiring magnets</td><td>FAUST</td><td></td><td>5 mm long-2 mm &amp;oslash;</td></tr><tr><td>Polystyrene round bottom tubes 5 ml</td><td>Falcon-BD Bioscience</td><td></td><td></td></tr><tr><td>Magnetic stirrer with heating function</td><td>IKA-RCT-standard</td><td>9720250</td><td></td></tr><tr><td>Petri dishes 100 mm, sterile</td><td>TPP</td><td>6223201</td><td></td></tr><tr><td>25 G needles</td><td>Terumo</td><td></td><td></td></tr><tr><td>anti mouse CD45 Ab</td><td>Biolegend</td><td></td><td>Clone 30-F11</td></tr><tr><td>anti mouse CD11c Ab</td><td>Biolegend</td><td></td><td>Clone N418</td></tr><tr><td>anti mouse Podoplanin Ab</td><td>Biolegend</td><td></td><td>Clone 8.1.1</td></tr><tr><td>anti mouse CD31 Ab</td><td>Biolegend</td><td></td><td>Clone MEC13.3</td></tr><tr><td>&lt;br/&gt; Eppendorf Xplorer plus, Multichannel</td><td>Eppendorf</td><td>4861 000.821/830</td><td>1.250 &amp;micro;l max. volume</td></tr><tr><td>anti mouse CD140a</td><td>Biolegend</td><td></td><td>Clone APA5</td></tr><tr><td>anti mouse CD80</td><td>Biolegend</td><td></td><td>Clone 16-10A1</td></tr><tr><td>anti mouse CD40</td><td>Biolegend</td><td></td><td>Clone 1C10</td></tr><tr><td>anti mouse I-Ab</td><td>Biolegend</td><td></td><td>Clone AF6-120.1</td></tr><tr><td>anti mouse CD274 (PD-L1)</td><td>Biolegend</td><td></td><td>Clone 10F.9G2</td></tr><tr><td>LIVE/DEAD Fixable Near-IR Dead Cell stain kit</td><td>Invitrogen</td><td>L10119</td><td></td></tr></tbody></table>

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.

O presente protocolo é um protocolo de digestão modificada publicada por ligação et al., 2007 6 com um menor tempo de digestão (45 minutos no máximo), devido à desagregação mecânica com uma pipeta multicanal automatizado. Além disso, o processo é mais padronizado, minimiza a degradação de marcadores de superfície em diferentes células do estroma de nódulos linfáticos e permite a manipulação de mais de uma amostra ao mesmo tempo. Colagenase IV e colagen...

Watch this Scientific Journal Video about Isolation of Murine Lymph Node Stromal Cells at JoVE.com

Isolation of Murine Lymph Node Stromal Cells

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 ...

isolation-of-murine-lymph-node-stromal-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

31.1K visualizações.  University of Basel and University Hospital Basel. 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. 

Vídeo: Isolation of Murine Lymph Node Stromal Cells

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolamento de células T CD4 + a partir dos nós linfáticos rato Usando Miltenyi Purificação MACS

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Biologia

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

O isolamento de linfócitos de rato Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Imunologia e Infecção

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Isolamento e Th17 Diferenciação de Linfócitos T CD4 Naïve

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

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, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Isolamento, identificação e purificação de células epiteliais do timo murino

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

Isolamento e Caracterização de citometria de fluxo de linfócitos de murino Intestino Delgado

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Isolamento e Activação de Linfócitos murinos

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Isolando os linfócitos de rato pequeno Intestinal sistema imunológico

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Otimizando o isolamento de células estromais do timo e da medula óssea

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

Autor em destaque: Avançando na pesquisa hematopoiética usando isolamento de células estromais para sequenciamento de células únicas

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

Isolamento de células estromais de órgãos hematopoiéticos

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

Isolamento de Murino Linfonodo células estromais

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