Les auteurs tiennent à remercier Dietrich à Marilyn à l&#39;installation de débit de base UGB Cytométrie pour son aide avec le tri cellulaire et Peter Mottram pour son aide avec les microphotographies. Ce travail a été financé par le Flight Attendant Institut de recherche médicale, le Prix du LSU-concurrentiel du Programme de recherche et les subventions du NIH / NIAID P20 RR020159 et R03AI081171.

1. Perfusion pulmonaire et la suspension seule cellule <ol><li> Euthanasier la souris avec une injection intrapéritonéale de kétamine / xylazine mélange anesthésique (en mg / souris kétamine 1.8, xylazine 0,19) et une exsanguination par la veine fémorale </li><li> Exposer la cavité thoracique en coupant et en tirant doucement en arrière la peau externe du péritoine. Procéder à l&#39;ouverture du diaphragme par la coupe de la cage thoracique afin d&#39;exposer à la fois le cœur et les poumons. </li><li> Perfuser délicatement les poumons en utilisant une seringue de 5 ml remplie avec de l&#39;EDTA-HBSS (1mM EDTA en calcium / magnésium sans HBSS). Connecter une aiguille de 25 G et insérer l&#39;aiguille dans le ventricule droit. Pour éviter les fuites lors de l&#39;injection, sécuriser le tissu myocardique autour de l&#39;aiguille en utilisant une pince. Doucement injecter la solution, tout en maintenant une pression constante. Perfusion précis se traduira par l&#39;inflation du poumon et un changement de couleur rose / blanc. </li><li> Retirer les ganglions lymphatiques drainant à écarter toute contamination éventuelle de ce tissu. </li><li> Recueillirtissu pulmonaire, transfert à une boîte de Pétri sur la glace, et le hacher en petits morceaux à l&#39;aide d&#39;une lame de rasoir. </li><li> Transfert des tissus à la terre dans un tube gentleMACS C contenant 5 ml / poumon de la solution de digestion par la collagénase (collagénase de type 1A 0,5 mg / ml, plus de type IV pancréatique bovine DNase 20 ug / ml dans du HBSS contenant 5% de FBS, 100 U / ml de pénicilline et 100 ug ml de streptomycine /). </li><li> Utilisez le dissociateur gentleMACS pour obtenir une suspension cellulaire unique. Choisissez le programme: m_lung_01. Incuber l&#39;échantillon à 37 ° C pendant 30 minutes. Agiter le tube toutes les 5 minutes pour remettre les fragments de tissus. Procéder à m_lung_02 programme. </li><li> Transférer l&#39;échantillon dans un tube de 15 ml conique et centrifuger pendant 10 minutes à 335 x g à 4 ° C. Après cette étape, gardez tous les réactifs et centrifugations à 4 ° C pour empêcher la réduction de la viabilité cellulaire. </li><li> Rejeter le surnageant et lyser les globules rouges restants en ajoutant 2 ml de tampon de lyse ACK pendant 1 minute à température ambiante. Laver les cellules avec 13ml de BSA PBS/0.5% froid et centrifuger pendant 10 minutes à 335 x g à 4 ° C. </li><li> Rejeter le surnageant, resuspendre nombre total de cellules dans 5 ml de BSA PBS/0.5% froid et passer un maillage complet 100 um en nylon. </li><li> Déterminer le nombre total de cellules en utilisant colorant exclusion du bleu trypan. </li></ol> 2. Isolation magnétique et CD11c + cellules d&#39;enrichissement <ol><li> Centrifuger la suspension seule cellule pendant 10 minutes à 200 x g à 4 ° C et éliminer le surnageant. </li><li> Resuspendre le culot cellulaire dans 400 pl de tampon de séparation (PBS, BSA 0,5%, et 2 mM d&#39;EDTA) par 10 8 cellules totales. Bloc-Fc des anticorps médiée imprécise de liaison en utilisant des anticorps anti-CD16/CD32 (0.5μg / 10 6 cellules) pendant 30 minutes à 4 ° C. </li><li> Laver les cellules avec 10 ml de tampon de séparation à froid et centrifuger pendant 10 minutes à 200 x g à 4 ° C. Resuspendre les cellules dans 400 ul de tampon de séparation. </li><li> Ajouter 100 ul de microbilles CD11c par 10 8 </soutien cellules&gt; total. Bien mélanger et incuber pendant 15 minutes au réfrigérateur (2-8 ° C). </li><li> Laver les cellules avec 10 ml de tampon de séparation à froid et centrifuger pendant 10 minutes à 200 x g à 4 ° C et éliminer le surnageant. </li><li> Reprendre le culot cellulaire dans 3 ml de tampon de séparation à froid. </li><li> Effectuer la séparation magnétique à l&#39;aide de la autoMACS Pro TM Séparateur en sélectionnant le posseld programme. Recueillir la fraction positive (0,5 ml). </li></ol> 3. Cellules dendritiques classiques (CDC) d&#39;isolation <ol><li> Incuber enrichi CD11c suspension cellulaire positifs avec des anti-CD11c PE-Cy7 et anti-IA/IE (CMH-II)-FITC anticorps pendant 30 minutes à 4 ° C. La concentration d&#39;anticorps est optimisée 0,5 pg / 10 6 de cellules. </li><li> Laver les cellules avec de la BSA PBS/0.5% froid et centrifuger pendant 10 minutes à 335 x g à 4 ° C. Resuspendre les cellules dans 0,5 ml de BSA PBS/0.5% et les faire passer à travers une maille de 40 um de nylon. </li><li> Passez àtrier les cellules en utilisant le trieur FACS Aria cellulaire dans le mode de pureté. Ajouter 100 ul de BSA PBS/0.5% sur les tubes de collecte afin de prévenir tout dommage des cellules purifiées. Gardez vos cellules à une température réfrigérée au long du processus de tri cellulaire. </li></ol> 4. Protocole alternatif pour l&#39;obtention d&#39;une suspension cellulaire unique et CD11c + cellules d&#39;enrichissement <ol><li> En remplacement de la dissociateur gentleMACS, le tissu pulmonaire à la terre de l&#39;étape 1.5 peut être transféré à un tube de 15 ml conique contenant 5 ml de solution de collagénase par poumon et incuber pendant 1 heure à 37 ° C. Vortex cellules toutes les 15 minutes afin de remettre en suspension des fragments de tissus. Perturber le tissu en passant l&#39;échantillon 6-8 fois dans et à travers une seringue de 3 ml connecté à une aiguille 20 G. Évitez de faire des bulles pendant le processus, sinon la viabilité des cellules sera compromise. Une fois la suspension cellulaire unique est obtenue, passez à l&#39;étape 1.8. </li><li> En remplacement de la séparation AutoMACSPro MCTor, isolation magnétique pour l&#39;enrichissement des cellules CD11c, peut également être effectuée par le passage de la suspension cellulaire manuelle à travers des colonnes MACS deux. La sélection des colonnes appropriées varieront en fonction de la taille de l&#39;échantillon. </li></ol> 5. Les résultats représentatifs: Lung CDC sont identifiés comme CD11c Salut / CMH-II population de cellules Salut. Comme indiqué dans la Fig. 1, CDC représentent un pourcentage inférieur de 1,4% par rapport à certains autres tissus tels que la rate (4,8%). Cependant, contrairement à la rate, les poumons des cellules CD11c positifs d&#39;exprimer des quantités différentes de CMH-II, y compris une population cellulaire différent de celui des CDC. Après la préparation à cellule unique, le rendement cellulaire des cellules pulmonaire totale était d&#39;environ 3,0 à 3,8 x 10 7 cellules / poumon avec une viabilité de 60-70% à partir de laquelle un peu plus de 1% a représenté sous la CDC. Ce pourcentage a augmenté après l&#39;isolement CD11c magnétique où le poumon totale CDC a été incrémenté environ 10 tIMES (&gt; 16%) de la préparation originale (figure 2A). Cependant, les cellules CD11c exprimer de faibles quantités de CMH-II (CD11c Salut / CMH-II lo, les macrophages) représentent une population de cellules contaminantes haute (&gt; 70%) qui a été mélangé avec le sous-ensemble des poumons CDC. Comme indiqué dans la Fig. 2B, ces cellules ont été éliminées après l&#39;étape de tri cellulaire lorsque la pureté CDC atteint&gt; 96%. Le rendement habituel de la CDC, après toute la procédure était de 5 x 10 4 cellules / poumon. Microphotographies montrent des caractéristiques morphologiques de la CDC comme des cellules arrondies avec les dendrites typiques (panneau supérieur). D&#39;autre part, CD11c Salut / CMH-II lo représenté un sous-ensemble morphologiquement différentes qui montre protubérances cellulaires qui sont typiques des macrophages (MΦ, panneau inférieur) 12. <img alt="Figure 1" src="/files/ftp_upload/3563/3563fig1.jpg" /> Figure 1. Différentiel DC fréquence des poumons de souris et la rate. Lung unetissu splénique ND de souris C57BL / 6 ont été collectées et traitées avec de la collagénase. Après digestion à la collagénase, les cellules ont été colorées avec anti-souris CD11c-PE-Cy7 et anti-souris IA / IE-FITC. Parcelles représentant de cytométrie en flux montrent des pourcentages de la CDC (CD11c Salut / CMH-II Salut) dans les poumons et la rate. <img alt="Figure 2" src="/files/ftp_upload/3563/3563fig2.jpg" /> Figure 2. Isolement de la CDC à partir des poumons de souris. Poumons de souris des cellules de suspension unique a été étiqueté avec des anti-CD11c microbilles et passé à travers un séparateur de cellules automatique. A) Un graphique représentatif montre les pourcentages de la CDC (CD11c Salut / CMH-II Salut) et Les macrophages (MΦ, CD11c Salut / CMH-II LO) des populations après enrichissement CD11c. Coloration plus de la fraction enrichie en utilisant suivi anti-souris CD11c-PE-Cy7 et anti-souris IA / IE-FITC. Double cellules positives ont été triées et analysées par cytométrie en flux. Pourd&#39;identifier leur morphologie, les cellules ont été cytospun et colorées avec une modification Wright-Giemsa coloration. B) parcelles représentant montrent des pourcentages de la CDC et MΦ après tri cellulaire. Microphotographies montrent une image représentative du poumon et de la CDC MΦ poumon. Barre d&#39;échelle = 20 um.

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Les auteurs n&#39;ont rien à révéler.

L&#39;isolement de la souris pulmonaires DC est une technique importante pour l&#39;étude d&#39;un large éventail de stimuli respiratoires. Le processus d&#39;obtention de ces cellules comprend des étapes critiques qui empêchent la perte de cellules ainsi que la viabilité des cellules et la pureté. Perfusant le poumon avant la collecte aidera à éliminer toutes les cellules périphériques ainsi que de réduire les érythrocytes contaminant. L&#39;utilisation de la dissociation peut être automatisé advanteogus ...

<table width="200" border="1"><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> Tampon de lyse ACK </td><td> Invitrogen </td><td> D6-0005DG </td><td></td></tr><tr><td> Anti-souris CD11c (HL3) </td><td> BD Pharmingen </td><td> 5580979 </td><td> PE-Cy7 conjugués </td></tr><tr><td> Anti-souris IA / IE (269) </td><td> BD-Pharmingen </td><td> 553623 </td><td> Conjugué au FITC </td></tr><tr><td> Collangenase type 1A </td><td> Sigma </td><td> 9891-500MG </td><td></td></tr><tr><td> Crépines cellulaire </td><td> BD Falcon </td><td> 352340, 352360 </td><td></td></tr><tr><td> CD11c (N418) Microbilles </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> Hank solution saline équilibrée </td><td> Invitrogen </td><td> 14170 </td><td></td></tr><tr><td> Solution tampon HEPES </td><td> Invitrogen </td><td> 15630 </td><td></td></tr><tr><td> Boîtes de Pétri de 60 mm </td><td> BD Falcon </td><td> 351016 </td><td></td></tr><tr><td> GentleMACS ™ C tubes </td><td> Miltenyi </td><td> 130-093-237 </td><td></td></tr><tr><td> Douceur dissociateur MACS </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

Cellules dendritiques pulmonaires (DC) jouent un rôle fondamental dans la détection des pathogènes envahisseurs 1,2 ainsi que dans le contrôle des réponses tolérogènes 3 dans les voies respiratoires. Au moins trois sous-ensembles principaux de cellules dendritiques pulmonaires ont été décrites chez la souris: DC conventionnelles (CDC) 4, DC plasmacytoïdes (pDC) 5 et le tueur de l&#39;IFN-production DC (IKDC) 6,7. Le sous-ensemble CDC est le sous-ensemble DC les plus éminents dans les poumons 8. Le marqueur commun connus pour identifier des sous-DC est CD11c, un type que je transmembranaire intégrines (β2) qui est également exprimé sur les monocytes, les macrophages, les neutrophiles et des cellules B 9. Dans certains tissus, en utilisant comme marqueur CD11c pour identifier la souris DC est valable, comme dans la rate, où la plupart des cellules CD11c + représentent le sous-ensemble des CDC, qui exprime des niveaux élevés de l&#39;complexe majeur d&#39;histocompatibilité de classe II (CMH-II). Toutefois, le poumon est un tissu plus hétérogène, où secôté des sous-ensembles DC, il ya un pourcentage élevé d&#39;une population de cellules distinctes qui exprime des niveaux élevés de CD11c bout de faibles niveaux de CMH-II. Basé sur sa caractérisation et surtout sur ​​l&#39;expression de ses F4/80, un marqueur des macrophages spléniques, le CD11c Salut CMH-II de la population lo cellules du poumon a été identifié comme les macrophages pulmonaires 10 et plus récemment, comme un précurseur potentiel DC 11. Contrairement à la souris PDC, l&#39;étude du rôle spécifique de la CDC dans la réponse immunitaire pulmonaire a été limitée en raison de l&#39;absence d&#39;un marqueur spécifique qui pourrait aider dans l&#39;isolement de ces cellules. Par conséquent, dans ce travail, nous décrivons une procédure pour isoler hautement purifiée de souris pulmonaires CDC. L&#39;isolement des sous-ensembles DC pulmonaires représente un outil très utile afin de mieux connaître la fonction de ces cellules en réponse à des agents pathogènes des voies respiratoires ainsi que les facteurs environnementaux qui peuvent déclencher la réponse immunitaire de l&#39;hôte dans les poumons.

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

Isolation of Mouse Lung Dendritic Cells

Une préparation hautement purifiée de cellules de souris pulmonaires dendritiques est décrite. Un accent particulier est donné à l&#39;isolement de sous-ensemble de cellules dendritiques conventionnelles.

L&#39;isolement des cellules de souris poumon dendritiques

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 vues.  Louisiana State University. Une préparation hautement purifiée de cellules de souris pulmonaires dendritiques est décrite. Un accent particulier est donné à l'isolement de sous-ensemble de cellules dendritiques conventionnelles.

Vidéo: 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.

L&#39;isolement des cellules de souris cavité péritonéale

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

Recherche

Immunologie et infection

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.

L&#39;isolement et la caractérisation des cellules dendritiques et les macrophages de l&#39;intestin de souris

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.

Isolement des cellules dendritiques myéloïdes et les cellules épithéliales du thymus humain

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.

Une méthode de rendement efficace et haute pour l&#39;isolement des souris dendritiques sous-ensembles cellulaires

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.

Fluorescence-tri cellulaire activé pour la purification de cellules dendritiques plasmacytoïdes de la moelle osseuse de souris

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.

Génération de cellules humaines dérivées de monocytes dendritiques du sang total

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 membrane externe Vésicules: Isolement et analyse de leur potentiel pro-inflammatoire sur Macrophages

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.

Human Cells Lung dendritiques: Répartition spatiale et phénotypique identification dans endobronchique Biopsies par immunohistochimie et cytométrie de flux

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.

coupe de précision de la souris Tranches Lung pour visualiser les cellules vivantes pulmonaire dendritiques

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.

Isolement de cellules dendritiques de l’appareil reproducteur féminin humain pour des études phénotypiques et fonctionnelles

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