Os autores gostariam de agradecer a Marilyn Dietrich no Centro de Núcleo LSU Citometria de Fluxo por sua ajuda com a separação de células e Mottram Peter por sua ajuda com as microfotografias. Este trabalho foi financiado pela Flight Attendant Medical Research Institute, da LSU-Competitiva Prêmio Programa de Pesquisa, e os subsídios NIH / NIAID P20 RR020159 e R03AI081171.

1. Perfusão pulmonar e suspensão única célula <ol><li> Euthanize o mouse com uma injeção intraperitoneal de cetamina / xilazina mistura anestésica (em mg / mouse ketamina 1.8, xilazina 0,19) e sangria pela veia femoral </li><li> Expor a cavidade torácica, cortando e puxando para trás a pele externa do peritônio. Proceder à abertura do diafragma, cortando a caixa torácica para expor tanto o coração e os pulmões. </li><li> Perfundir gentilmente os pulmões com uma seringa de 5 ml preenchido com EDTA-HBSS (1mM EDTA de cálcio / magnésio livre de HBSS). Conectar uma agulha G 25 e inserir a agulha para o ventrículo direito. Para evitar fugas durante a injeção, segura o tecido ao redor da agulha myocardiac usando uma pinça. Suavemente injectar a solução, mantendo uma pressão constante. Perfusão será a precisão do resultado da inflação do pulmão e uma mudança de cor de rosa / branco. </li><li> Remover linfonodos drenantes para descartar qualquer possível contaminação a partir deste tecido. </li><li> Coletartecido pulmonar, a transferência para uma placa de Petri sobre gelo, e pique em pedaços pequenos usando uma lâmina de barbear. </li><li> Transferência de tecidos fundada em um tubo C gentleMACS contendo 5 ml / pulmão de solução de digestão da colagenase (colagenase tipo 1A 0,5 mg / ml, mais tipo IV de pâncreas bovino DNase 20 mg / ml em HBSS contendo 5% de FBS, 100 U / ml de penicilina e 100 mg / estreptomicina ml). </li><li> Use o dissociator gentleMACS para obter uma suspensão única célula. Escolha o programa: m_lung_01. Incubar amostra a 37 ° C por 30 minutos. Agite tubo a cada 5 minutos para voltar a suspender fragmentos de tecido. Prosseguir com m_lung_02 programa. </li><li> Transferência de amostra para um tubo cônico de 15 ml e centrifugar por 10 minutos a 335 x g a 4 ° C. Após esta etapa, manter todos os reagentes e centrifugação a 4 ° C para evitar a redução da viabilidade celular. </li><li> Elimine o sobrenadante e lise restantes células vermelhas do sangue, adicionando 2 ml de tampão de lise ACK por 1 minuto à temperatura ambiente. Lavar as células com 13ml de BSA PBS/0.5% fria e centrifugação por 10 minutos a 335 x g a 4 ° C. </li><li> Elimine o sobrenadante, ressuspender total de células em 5 ml de BSA PBS/0.5% fria e passe completo de 100 M malha de nylon. </li><li> Determinar o número total de células usando corante de exclusão de tripan blue. </li></ol> 2. Isolamento magnético e CD11c + enriquecimento de células <ol><li> Centrífuga suspensão única célula por 10 minutos a 200 x g a 4 ° C e descartar o sobrenadante. </li><li> Ressuspender pellet celular em 400 mL de tampão de separação (PBS, BSA 0,5%, e 2 mM de EDTA) por 10 8 células total. Bloco de anticorpos Fc mediada inespecíficos ligação usando anti-CD16/CD32 anticorpos (0.5μg / 10 6 células) por 30 minutos a 4 ° C. </li><li> Lavar as células com 10 ml de tampão de separação fria e centrifugação por 10 minutos a 200 x g a 4 ° C. Ressuspender as células em 400 mL de tampão de separação. </li><li> Adicione 100 ml de microesferas de CD11c por 10 8 </sup&gt; células total. Misture bem e incubar por 15 minutos na geladeira (2-8 ° C). </li><li> Lavar as células com 10 ml de tampão de separação fria e centrifugação por 10 minutos a 200 x g a 4 ° C e descartar o sobrenadante. </li><li> Ressuspender o pellet celular em 3 ml de tampão de separação frio. </li><li> Realizar separação magnética usando o separador autoMACS Pro TM, selecionando o posseld programa. Recolher a fração positiva (0,5 ml). </li></ol> 3. Células dendríticas convencional (CDC) de isolamento <ol><li> Incubar enriquecido CD11c suspensão de células positivas com anti-CD11c-PE Cy7 e anti-IA/IE (MHC-II)-FITC anticorpos durante 30 minutos a 4 ° C. Concentração otimizada de anticorpos é de 0,5 mg / 10 6 de células. </li><li> Lavar as células com BSA PBS/0.5% fria e centrifugação por 10 minutos a 335 x g a 4 ° C. Ressuspender as células em 0,5 ml de BSA PBS/0.5% e passá-las através de uma malha de nylon 40 mM. </li><li> Proceder àclassificar as células usando o classificador de células FACS Aria no modo de pureza. Adicione 100 ml de BSA PBS/0.5% para os tubos de coleta, a fim de evitar danos de células purificadas. Mantenha suas células, a uma temperatura de refrigeração em todo o processo de separação de células. </li></ol> 4. Protocolo alternativo para a obtenção de uma suspensão única célula e CD11c + enriquecimento de células <ol><li> Em substituição do dissociator gentleMACS, tecido pulmonar fundamentada a partir do passo 1.5 pode ser transferida para um tubo cônico de 15 ml contendo 5 ml de solução de colagenase por pulmão e incubar por 1 hora a 37 ° C. Vortex células a cada 15 minutos, a fim de ressuspender fragmentos de tecido. Perturbar o tecido, passando a amostra 6-8 vezes dentro e para fora através de uma seringa de 3 ml conectada a uma agulha G 20. Evite fazer bolhas durante o processo, caso contrário, a viabilidade celular será comprometida. Uma vez que a suspensão única célula é obtida, continue para o passo 1.8. </li><li> Em substituição do TM AutoMACSPro separaçãotor, o isolamento magnético para o enriquecimento das células CD11c, também pode ser realizada pela passagem da suspensão de células manualmente através de duas colunas MACS. A seleção das colunas apropriado irá variar dependendo do tamanho da amostra. </li></ol> 5. Resultados representativos: Pulmão CDC são identificados como CD11c oi / MHC-II população de células oi. Como mostrado na figura. 1, do CDC representam uma porcentagem menor de 1,4% quando comparado a alguns outros tecidos, como baço (4,8%). No entanto, em contraste com o baço, as células do pulmão CD11c positiva expressar diferentes quantidades de MHC-II, incluindo uma população de células diferentes do que a de CDC. Após a preparação de uma única célula, o rendimento das células pulmonar total foi de cerca de 3,0-3,8 x 10 7 células / pulmão com uma viabilidade de 60-70% dos quais pouco mais de 1%, representado subconjunto CDC. Esta percentagem foi aumentada após o isolamento CD11c magnética onde o total de pulmão CDC foi incrementado cerca de 10 times (&gt; 16%) desde a preparação original (Fig. 2A). No entanto, as células CD11c expressar baixas quantidades de MHC-II (CD11c oi / MHC-II eis que macrófagos) representou uma população de células de alta contaminação (&gt; 70%), que foi misturado com o subconjunto CDC pulmão. Como mostrado na figura. 2B, essas células foram eliminados após a etapa de separação de células, quando a pureza do CDC chegou a&gt; 96%. O rendimento habitual do CDC, após todo o procedimento foi de 5 x 10 4 células / pulmão. Microfotografias mostram características morfológicas do CDC como células arredondadas com as dendrites típicas (painel superior). Por outro lado, CD11c oi / MHC-II representaram um subconjunto lo morfologicamente diferentes que mostra protuberâncias celulares que são típicas de macrófagos (MΦ painel, menor) 12. <img alt="Figura 1" src="/files/ftp_upload/3563/3563fig1.jpg" /> Figura 1. Diferencial de freqüência DC no pulmão do rato e do baço. Um pulmãond tecido do baço de camundongos C57BL / 6 foram coletados e tratados com colagenase. Após digestão com colagenase, as células foram marcadas com anti-mouse CD11c-PE-Cy7 e anti-mouse IA / IE-FITC. Parcelas representativas de citometria de fluxo mostram percentuais de CDC (CD11c oi / MHC-II oi) no pulmão e baço. <img alt="Figura 2" src="/files/ftp_upload/3563/3563fig2.jpg" /> Figura 2. Isolamento de CDC do pulmão do rato. Rato pulmão suspensão única célula foi marcado com anti-CD11c micro e passou por um separador automático de células. A) Um representante trama mostra os percentuais de CDC (CD11c oi / MHC-II oi) e macrófagos (MΦ, CD11c oi / MHC-II lo) após o enriquecimento CD11c populações. Coloração mais da fração enriquecida seguido usando anti-mouse CD11c-PE-Cy7 e anti-mouse IA / IE-FITC. Duplo células positivas foram classificadas e analisadas por citometria de fluxo. Paraidentificar sua morfologia, as células foram cytospun e marcadas com uma coloração modificada Wright-Giemsa. B) parcelas Representante mostram percentuais de CDC e MΦ após separação de células. Microfotografias mostram uma imagem representativa do pulmão CDC e MΦ pulmão. Barra de escala = 20 mM.

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Isolamento de pulmonar do rato DC é uma técnica importante para o estudo de uma ampla gama de estímulos respiratórios. O processo de obtenção dessas células inclui passos críticos que impedem a perda de células, bem como a viabilidade celular e pureza. Perfusão do pulmão antes da coleta ajudará a eliminar as células periféricas, bem como reduzir os eritrócitos contaminantes. O uso da dissociação automatizado pode ser advanteogus quando um grande número de pulmões são tratados, caso contrário, você ...

<table width="200" border="1"><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comments (opcional) </td></tr><tr><td> Tampão de lise ACK </td><td> Invitrogen </td><td> D6-0005DG </td><td></td></tr><tr><td> Anti-camundongo CD11c (HL3) </td><td> BD PharMingen </td><td> 5580979 </td><td> PE-Cy7 conjugados </td></tr><tr><td> Anti-camundongo IA / IE (269) </td><td> BD-PharMingen </td><td> 553623 </td><td> FITC conjugado </td></tr><tr><td> Collangenase tipo 1A </td><td> Sigma </td><td> 9891-500mg </td><td></td></tr><tr><td> Filtros celular </td><td> BD Falcon </td><td> 352340, 352360 </td><td></td></tr><tr><td> CD11c (N418) microesferas </td><td> Miltenyi </td><td> 130-052-001 </td><td></td></tr><tr><td> DNase I </td><td> Sigma </td><td> D5025-150KU </td><td></td></tr><tr><td> Solução salina balanceada de Hank </td><td> Invitrogen </td><td> 14170 </td><td></td></tr><tr><td> Solução tampão Hepes </td><td> Invitrogen </td><td> 15630 </td><td></td></tr><tr><td> Placas de Petri 60 milímetros </td><td> BD Falcon </td><td> 351016 </td><td></td></tr><tr><td> GentleMACS ™ C tubos </td><td> Miltenyi </td><td> 130-093-237 </td><td></td></tr><tr><td> Dissociator MACS gentil </td><td> Miltentyi </td><td> 130-093-235 </td><td></td></tr><tr><td> AutoMACS-Pro ™ </td><td> Miltenyi </td><td> 130-092-545 </td><td></td></tr><tr><td> FASCS Aria </td><td> BD </td><td></td><td></td></tr></table>

isolation of mouse lung dendritic cells

Células pulmonares dendríticas (DC) desempenham um papel fundamental na detecção invadindo 1,2 patógenos, bem como no controle de respostas tolerogênico 3 no trato respiratório. Pelo menos três subconjuntos principais de células dendríticas pulmonares têm sido descritas em ratos: DC convencional (CDC) 4, plasmocitóide DC (PDC) 5 e IFN-produzindo assassino DC (IKDC) 6,7. O subconjunto do CDC é o subconjunto DC mais proeminentes no pulmão 8. O marcador comum conhecida para identificar subgrupos DC CD11c é, um tipo que eu transmembrana integrina (β2), que também é expresso em monócitos, macrófagos, neutrófilos e algumas células B 9. Em alguns tecidos, usando CD11c como um marcador para identificar rato DC é válido, como no baço, onde a maioria das células CD11c + representa o subconjunto do CDC que expressa altos níveis do complexo principal de histocompatibilidade classe II (MHC-II). No entanto, o pulmão é um tecido mais heterogêneo, onde sesubconjuntos lado DC, há uma elevada percentagem de uma população de células distintas que expressa altos níveis de CD11c combate os baixos níveis de MHC-II. Com base na sua caracterização e principalmente em sua expressão de F4/80, um marcador de macrófagos do baço, o CD11c oi MHC-II eis população de células do pulmão foi identificado como macrófagos pulmonares 10 e, mais recentemente, como um precursor potencial DC 11. Em contraste com o rato pDC, o estudo do papel específico do CDC na resposta imunológica pulmonar tem sido limitado devido à falta de um marcador específico que poderia ajudar no isolamento dessas células. Portanto, neste trabalho, descrevemos um procedimento para isolar altamente purificado do mouse pulmão CDC. O isolamento de subconjuntos pulmonar DC representa uma ferramenta muito útil para obter insights sobre a função destas células em resposta a patógenos respiratórios, bem como fatores ambientais que podem desencadear a resposta imune do hospedeiro no pulmão.

Watch this Scientific Journal Video about Isolation of Mouse Lung Dendritic Cells at JoVE.com

Isolation of Mouse Lung Dendritic Cells

A preparação altamente purificada de células dendríticas pulmonares do rato é descrito. Ênfase específica é dada para o isolamento de um subconjunto de células dendríticas convencionais.

Isolamento de células dendríticas pulmonares do rato

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Research

JoVE Journal

Immunology and Infection

28.9K visualizações.  Louisiana State University. A preparação altamente purificada de células dendríticas pulmonares do rato é descrito. Ênfase específica é dada para o isolamento de um subconjunto de células dendríticas convencionais.

Vídeo: Isolation of Mouse Lung Dendritic Cells

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

Isolamento de células de camundongo Peritoneal Cavity

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Imunologia e Infecção

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Isolamento e caracterização de células dendríticas e macrófagos a partir do intestino do rato

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

O isolamento de células dendríticas mielóides e células epiteliais de timo humano

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Um método de rendimento eficiente e alta para o isolamento de subpopulações de células dendríticas Rato

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Classificação de células activadas por fluorescência Para a purificação de células dendríticas plasmocitï¿½des do rato de Medula Óssea

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Geração de células dendríticas derivadas de monócitos humanos a partir de sangue inteiro

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Legionella pneumophila Outer Membrane vesículas: Isolamento e Análise do seu potencial pró-inflamatórios em macrófagos

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

Células Humanas Lung dendríticas: distribuição espacial e fenotípica Identificação em endobrônquica Biópsias imunohistoquímica e citometria de fluxo

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Rato pulmão Slices para Visualize células vivas pulmonar dendríticas precisão de corte

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

Isolamento de células dendríticas do tracto reprodutivo feminino humano para estudos fenotípica e funcionais

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...