Die Autoren möchten sich an Marilyn Dietrich an der LSU Durchflusszytometrie Core Facility danken für ihre Hilfe bei der Zellsortierung und Peter Mottram für seine Unterstützung bei der Mikrophotographien. Diese Arbeit wurde von der Flight Attendant Medical Research Institute, die LSU-Competitive Research Program Award und den NIH / NIAID Grants P20 RR020159 und R03AI081171 finanziert.

1. Lung Perfusion und einzigen Zellsuspension <ol><li> Euthanize die Maus mit einer intraperitonealen Injektion von Ketamin / Xylazin Narkose-Gemisch (in mg / Maus Ketamin 1,8, Xylazin 0,19) und Ausbluten über die Vena femoralis </li><li> Expose der Brusthöhle durch Schneiden und vorsichtig wieder die Außenhaut des Bauchfells. Fahren Sie mit dem Zwerchfell, indem die Rippen, um sowohl die Herz und Lunge aussetzen zu öffnen. </li><li> Perfundieren sanft die Lungen mit einer 5 ml Spritze mit EDTA-HBSS (1mM EDTA in Calcium / Magnesium-freien HBSS) gefüllt. Schließen Sie eine 25 G Nadel und legen Nadel in die rechte Herzkammer. Zur Vermeidung von Leckagen während der Injektion, sichern Sie die myokardiale Gewebe um die Nadel mit einer Pinzette. Vorsichtig injizieren Lösung bei konstantem Druck. Genaue Perfusion wird in Lunge Inflation und ein Farbumschlag nach rosa / weiß Ergebnis. </li><li> Entfernen Lymphknoten um eine mögliche Verunreinigung von diesem Gewebe zu verwerfen. </li><li> SammelnLungengewebe, in eine Petrischale übertragen auf Eis, und hacken sie in kleine Stücke mit einer Rasierklinge. </li><li> Transfer geerdet Gewebe in eine gentleMACS C Rohr mit 5 ml / Lunge Kollagenaseverdau Lösung (Kollagenase Typ 1A 0,5 mg / ml plus Typ IV Rinderpankreas DNase 20 pg / ml in HBSS mit 5% FBS, 100 U / ml Penicillin und 100 ug / ml Streptomycin). </li><li> Verwenden Sie die gentleMACS dissociator auf eine einzelne Zelle Suspension zu erhalten. Wählen Sie Programm: m_lung_01. Inkubieren Sie die Probe bei 37 ° C für 30 Minuten. Schütteln Rohr alle 5 Minuten, um Gewebeteile zu resuspendieren. Fahren Sie mit dem Programm m_lung_02. </li><li> Transfer-Probe auf eine 15 ml konischen Röhrchen und zentrifugieren Sie für 10 Minuten bei 335 x g bei 4 ° C. Nach diesem Schritt zu halten alle Reagenzien und Zentrifugation bei 4 ° C zu einer Verringerung der Lebensfähigkeit der Zellen zu verhindern. </li><li> Überstand verwerfen und zu lysieren restlichen roten Blutkörperchen durch Zugabe von 2 ml ACK Lysepuffer für 1 Minute bei Raumtemperatur. Wash-Zellen mit 13ml kaltem PBS/0.5% BSA und Zentrifuge für 10 Minuten bei 335 x g bei 4 ° C. </li><li> Überstand resuspendieren gesamten Zellen in 5 ml kaltem PBS/0.5% BSA und pass gründliche einer 100 um Nylon-Mesh. </li><li> Bestimmen Sie die gesamte Zellzahl durch Trypanblau-Ausschluss-Farbstoff. </li></ol> 2. Magnetische Trennung und CD11c + Zellen Bereicherung <ol><li> Centrifuge einzigen Zellsuspension für 10 Minuten bei 200 x g bei 4 ° C und Überstand verwerfen. </li><li> Resuspendieren Zellpellet in 400 ul der Trennung Puffer (PBS, 0,5% BSA und 2 mM EDTA) pro 10 8 insgesamt Zellen. Block Fc-vermittelte unspezifische Antikörper-Bindung durch anti-CD16/CD32 Antikörper (0.5μg / 10 6 Zellen) für 30 Minuten bei 4 ° C. </li><li> Wash-Zellen mit 10 ml kaltem Trennpuffers und Zentrifuge für 10 Minuten bei 200 x g bei 4 ° C. Die Zellen in 400 ul der Trennung Puffer. </li><li> 100 l von CD11c MicroBeads pro 10 8 </sup&gt; insgesamt Zellen. Gut mischen und inkubieren für 15 Minuten in den Kühlschrank (2-8 ° C). </li><li> Wash-Zellen mit 10 ml kaltem Trennpuffers und Zentrifuge für 10 Minuten bei 200 x g bei 4 ° C und Überstand verwerfen. </li><li> Zellpellet in 3 ml kaltem Trennpuffers. </li><li> Führen magnetische Trennung mit dem autoMACS Pro TM Separator, indem Sie das Programm posseld. Sammeln Sie die positiven Fraktion (0,5 ml). </li></ol> 3. Konventionelle dendritischen Zellen (CDC) Isolierung <ol><li> Inkubieren bereichert CD11c positive Zellsuspension mit anti-CD11c PE-Cy7 und anti-IA/IE (MHC-II)-FITC-Antikörper für 30 Minuten bei 4 ° C. Optimierte Konzentration von Antikörpern liegt bei 0,5 pg / 10 6 Zellen. </li><li> Wash-Zellen mit kaltem PBS/0.5% BSA und Zentrifuge für 10 Minuten bei 335 x g bei 4 ° C. Die Zellen in 0,5 ml PBS/0.5% BSA und leiten sie durch ein 40 um Nylon-Mesh. </li><li> Gehen Sie zurArt der Zellen mit Hilfe der FACS Aria Zellsortierer in der Reinheit Modus. 100 l des PBS/0.5% BSA, um die Sammlung Röhren, um eine Beschädigung der gereinigten Zellen verhindern. Halten Sie Ihre Zellen bei einer Kühltemperatur in der Zelle Sortierung. </li></ol> 4. Alternate-Protokoll für den Erhalt einer Zellsuspension und CD11c + Zellen Bereicherung <ol><li> Als Ersatz für die gentleMACS dissociator kann geerdet Lungengewebe aus Schritt 1.5 in ein 15 ml konischen Röhrchen mit 5 ml Kollagenase-Lösung pro Lunge und inkubieren für 1 Stunde bei 37 ° C übertragen werden Vortex-Zellen alle 15 Minuten, um Gewebeteile zu resuspendieren. Stören des Gewebes, indem die Probe 6-8 mal in und durch eine 3 ml Spritze verbunden, um eine 20 G Nadel. Vermeiden Sie Luftblasen während des Prozesses, da sonst die Lebensfähigkeit der Zellen beeinträchtigt wird. Sobald einzelne Zelle Suspension erhalten wird, weiter auf 1,8 Schritt. </li><li> Als Ersatz für die AutoMACSPro TM Trennungtor, magnetische Trennung für die Anreicherung der CD11c Zellen, kann auch durch das Bestehen der Zellsuspension manuell durch zwei MACS Säulen durchgeführt werden. Die Auswahl der entsprechenden Spalten ist abhängig von der Stichprobengröße. </li></ol> 5. Repräsentative Ergebnisse: Lung CDC als CD11c hallo / MHC-II hallo Zellpopulation identifiziert. Wie in Abb.. 1, vertreten cDC einen kleineren Prozentsatz von 1,4%, wenn zu einigen anderen Geweben wie Milz (4,8%) verglichen. Doch im Gegensatz zu Milz-, Express-Lungen-CD11c positive Zellen unterschiedliche Mengen an MHC-II, einschließlich einer Zellpopulation, anders als das der CDC. Nach der Single-Cell-Vorbereitung, wurde die Zelle Ausbeute von insgesamt Lungenzellen etwa 3,0 bis 3,8 x 10 7 Zellen / Lunge mit einer Tragfähigkeit von 60-70% aus, die ein wenig mehr als 1% cDC Teilmenge vertreten. Dieser Prozentsatz wurde nach der CD11c magnetische Trennung, wo die gesamte Lunge cDC ca. 10 t erhöht wurde erhöhtimes (&gt; 16%) aus dem ursprünglichen Präparat (Abb. 2A). Allerdings vertreten CD11c exprimieren geringe Mengen an MHC-II (CD11c hallo / MHC-II lo, Makrophagen) eine hohe Kontamination Zellpopulation (&gt; 70%), die mit der Lunge cDC Teilmenge gemischt wurde. Wie in Abb.. 2B, wurden diese Zellen nach der Zellsortierung Schritt bei der CDC Reinheit&gt; 96% erreicht eliminiert. Die übliche Rendite von CDC nach dem ganzen Verfahren wurde 5 x 10 4 Zellen / Lunge. Mikrophotographien zeigen morphologische Merkmale der CDC als abgerundete Zellen mit den typischen Dendriten (oberes Bild). Auf der anderen Seite, vertreten CD11c hallo / MHC-II lo eine morphologisch unterschiedliche Teilmenge, dass zelluläre Ausstülpungen, die typisch für Makrophagen (MΦ, unten) 12 sind zeigt. <img alt="Abbildung 1" src="/files/ftp_upload/3563/3563fig1.jpg" /> Abbildung 1. Differential DC Frequenz in Maus Lunge und Milz. Lunge einend Milzgewebe von C57BL / 6 Mäuse wurden gesammelt und mit Kollagenase behandelt. Nach Kollagenaseverdau wurden die Zellen mit anti-Maus-CD11c-PE-Cy7 und anti-Maus-IA / IE-FITC gefärbt. Vertreter der Durchflusszytometrie Plots zeigen Prozentsätze der CDC (CD11c hallo / MHC-II hallo) in Lunge und Milz. <img alt="Abbildung 2" src="/files/ftp_upload/3563/3563fig2.jpg" /> Abbildung 2. Isolation von CDC aus Mauslunge. Mauslunge einzigen Zellsuspension wurde mit anti-CD11c Mikroperlen gekennzeichnet und durch eine automatische Zellseparator. A) Eine repräsentative Darstellung zeigt die Prozentsätze der CDC (CD11c hallo / MHC-II hallo) und Makrophagen (MΦ, CD11c hallo / MHC-II lo) Bevölkerung nach CD11c Bereicherung. Weitere Färbung der angereicherten Fraktion folgte mit Anti-Maus-CD11c-PE-Cy7 und anti-Maus-IA / IE-FITC. Doppel-positive Zellen wurden sortiert und mittels Durchflusszytometrie analysiert. ZuIdentifizierung ihrer Morphologie, Zellen wurden cytospun und mit einem modifizierten Wright-Giemsa-Färbung gefärbt. B) Repräsentative Plots zeigen Prozentsätze von CDC und MΦ nach cell sorting. Mikrofotografien zeigen ein repräsentatives Bild der Lunge cDC und Lunge MΦ. Balken = 20 um.

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Die Autoren haben nichts zu offenbaren.

Isolation der pulmonalen Maus DC ist eine wichtige Technik für die Untersuchung einer breiten Palette von Atemwegs-Reize. Das Verfahren zur Erlangung dieser Zellen enthält kritische Schritte, dass der Verlust von Zellen sowie die Lebensfähigkeit der Zellen und Reinheit zu verhindern. Perfusion der Lunge vor der Abholung wird dazu beitragen, die peripheren Zellen zu eliminieren, sowie verringern Verunreinigungen Erythrozyten. Der Einsatz der automatisierten Dissoziation kann advanteogus werden, wenn eine große Zahl v...

<table width="200" border="1"><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> ACK Lysepuffer </td><td> Invitrogen </td><td> D6-0005DG </td><td></td></tr><tr><td> Anti-Maus-CD11c (HL3) </td><td> BD Pharmingen </td><td> 5580979 </td><td> PE-Cy7 konjugierte </td></tr><tr><td> Anti-Maus-IA / IE (269) </td><td> BD-Pharmingen </td><td> 553623 </td><td> FITC-konjugierten </td></tr><tr><td> Collangenase Typ 1A </td><td> Sigma </td><td> 9891-500MG </td><td></td></tr><tr><td> Zell-Siebe </td><td> BD Falcon </td><td> 352340, 352360 </td><td></td></tr><tr><td> CD11c (N418) Microbeads </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> Hanks Balanced Salt Lösung </td><td> Invitrogen </td><td> 14170 </td><td></td></tr><tr><td> Hepes-Pufferlösung </td><td> Invitrogen </td><td> 15630 </td><td></td></tr><tr><td> Petrischalen 60 mm </td><td> BD Falcon </td><td> 351016 </td><td></td></tr><tr><td> GentleMACS ™ C Rohren </td><td> Miltenyi </td><td> 130-093-237 </td><td></td></tr><tr><td> Gentle MACS dissociator </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

Lung dendritische Zellen (DC) spielen eine fundamentale Rolle in der Sensorik eindringende Krankheitserreger 1,2 sowie in der Kontrolle von tolerogenen Antworten 3 in die Atemwege. Mindestens drei Teilmengen von Lungen-dendritische Zellen in Mäusen beschrieben worden: konventionelle DC (CDC) 4, plasmazytoide DC (pDC) 5 und die IFN-Produktion Killer DC (IKDC) 6,7. Die CDC Subset ist das prominenteste DC Teilmenge in der Lunge 8. Die gemeinsame Marker bekannt, DC Teilmengen zu identifizieren ist CD11c, eine Typ-I-Transmembran-Integrin (β2), die auch auf Monozyten, Makrophagen, Neutrophilen und einigen B-Zellen 9 ist zum Ausdruck gebracht. In einigen Geweben, mit CD11c als Marker zur Maus DC identifizieren gilt, wie in der Milz, wo die meisten CD11c + Zellen stellen die CDC Subset, das hohe Niveau des Haupthistokompatibilitätskomplex Klasse II (MHC-II) zum Ausdruck bringt. Allerdings ist die Lunge ein heterogenes Gewebe, wo seinSeite DC Teilmengen gibt es einen hohen Prozentsatz von einer deutlichen Zellpopulation, die ein hohes Maß an CD11c drückt Kampf geringe MHC-II. Basierend auf der Charakterisierung und vor allem auf seinen Ausdruck F4/80, eine Milz-Makrophagen-Marker, die CD11c hallo MHC-II lo Lunge Zellpopulation Lungenmakrophagen 10 und in jüngerer Zeit identifiziert worden, wie ein potenzieller DC Vorstufe 11. Im Gegensatz zu Maus pDC, hat das Studium der spezifischen Rolle der CDC in den pulmonalen Immunantwort wurde durch das Fehlen einer spezifischen Markierung, die in die Isolation dieser Zellen helfen könnte begrenzt. Daher wird in dieser Arbeit beschreiben wir ein Verfahren zu hoch gereinigten Mauslunge cDC isolieren. Die Isolierung der pulmonalen DC Teilmengen stellt ein sehr nützliches Werkzeug, um Einblicke in die Funktion dieser Zellen in Reaktion auf die Erreger respiratorischer Erkrankungen sowie Umwelt-Faktoren, die die Immunantwort des Wirtes in der Lunge auslösen kann gewinnen.

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

Isolation of Mouse Lung Dendritic Cells

Ein hoch gereinigte Präparation von Mauslunge dendritischen Zellen beschrieben wird. Besonderer Nachdruck wird auf die Isolierung von konventionellen dendritischen Zellen Teilmenge gegeben.

Isolierung von Maus Lung Dendritischen Zellen

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 Aufrufe.  Louisiana State University. Ein hoch gereinigte Präparation von Mauslunge dendritischen Zellen beschrieben wird. Besonderer Nachdruck wird auf die Isolierung von konventionellen dendritischen Zellen Teilmenge gegeben.

Serie: 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.

Isolierung von Maus Bauchhöhle 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 ...

Forschung

Immunologie und Infektion

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.

Isolierung und Charakterisierung von dendritischen Zellen und Makrophagen aus dem Mausdarm

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.

Isolierung von myeloider dendritischer Zellen und Epithelzellen Menschliche 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 ...

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.

Eine effiziente und High-Yield-Methode zur Isolierung von Maus über dendritische Zellen Subsets

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.

Fluoreszenz-aktivierte Zellsortierung zur Reinigung von Plasmazytoide dendritischer Zellen aus dem Knochenmark der Maus

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.

Herstellung von menschlichen Monozyten abgeleiteten Dendritischen Zellen aus Vollblut

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 der äußeren Membran - Vesikeln: Isolierung und Analyse ihrer Pro-inflammatorische Potential auf Makrophagen

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.

Menschliche Lunge Dendritische Zellen: Räumliche Verteilung und phänotypische Identifizierung in Endobronchial Biopsien mittels Immunhistochemie und Durchflusszytometrie

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.

Precision-cut Maus Lung Slices Live-Pulmonary Dendritische Zellen sichtbar zu machen

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.

Isolation von dendritischen Zellen aus menschlichen weiblichen Fortpflanzungsorgane für phänotypische und funktionelle Studien

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