Gli autori desiderano ringraziare per Marilyn Dietrich presso l&#39;impianto di core LSU Citometria a flusso per il suo aiuto con la separazione delle cellule e Peter Mottram per la sua assistenza con il microfotografie. Questo lavoro è stato finanziato dal Assistente di volo Medical Research Institute, l&#39;LSU-competitivi Premio programma di ricerca, e il NIH / NIAID Borse P20 RR020159 e R03AI081171.

1. Perfusione polmonare e sospensione singola cellula <ol><li> Euthanize il mouse con un&#39;iniezione intraperitoneale di ketamina / xylazina miscela anestetico (in mg / mouse ketamina 1,8, xilazina 0,19) e dissanguamento attraverso la vena femorale </li><li> Esporre la cavità toracica da taglio e delicatamente tirando indietro la pelle esterna del peritoneo. Procedere per aprire il diaframma tagliando la gabbia toracica per esporre sia il cuore ei polmoni. </li><li> Profumato delicatamente i polmoni usando una siringa da 5 ml riempito con EDTA-HBSS (1 mM EDTA di calcio / magnesio senza HBSS). Collegare un 25 ago G e inserire l&#39;ago nel ventricolo destro. Per evitare perdite durante l&#39;iniezione, fissare il tessuto miocardio intorno all&#39;ago utilizzando pinze. Delicatamente iniettare la soluzione, pur mantenendo una pressione costante. Perfusione accurata si tradurrà in inflazione al polmone e un cambiamento di colore rosa / bianco. </li><li> Rimuovere linfonodo drenante per eliminare qualsiasi potenziale contaminazione da questo tessuto. </li><li> Raccoglieretessuto polmonare, trasferimento in una capsula di Petri sul ghiaccio, e tagliare a piccoli pezzi con una lama di rasoio. </li><li> Trasferimento di tessuto a terra in un tubo gentleMACS C contenente 5 ml / polmone di soluzione collagenasi digestione (collagenasi tipo 1A 0,5 mg / ml, più di tipo IV bovina pancreatica DNasi 20 mg / ml in HBSS contenente il 5% FBS, 100 U / ml di penicillina e 100 mcg / ml streptomicina). </li><li> Utilizzare il dissociatore gentleMACS per ottenere una sospensione singola cella. Scegli il programma: m_lung_01. Incubare campione a 37 ° C per 30 minuti. Agitare tubo ogni 5 minuti per risospendere frammenti di tessuto. Procedere con m_lung_02 programma. </li><li> Trasferimento del campione ad un tubo da 15 ml e centrifugare per 10 minuti a 335 x g a 4 ° C. Dopo questo passo, conservare tutti i reagenti e centrifugazioni a 4 ° C per evitare una riduzione della vitalità delle cellule. </li><li> Gettare il surnatante e lisare restanti globuli rossi con l&#39;aggiunta di 2 ml di tampone di lisi ACK per 1 minuto a temperatura ambiente. Lavare le cellule con 13ml di BSA freddo PBS/0.5% e centrifugare per 10 minuti a 335 x g a 4 ° C. </li><li> Gettare il surnatante, risospendere le cellule totale in 5 ml di BSA freddo PBS/0.5% e passare un approfondito e 100 mesh di nylon micron. </li><li> Determinare il numero totale delle cellule utilizzando trypan esclusione colorante blu. </li></ol> 2. Isolamento magnetico e CD11c + arricchimento delle cellule <ol><li> Centrifugare sospensione singola cella per 10 minuti a 200 g a 4 ° C e scartare il surnatante. </li><li> Risospendere il pellet cellulare in 400 ml di tampone di separazione (PBS, 0,5% di BSA, e 2 mM EDTA) per 10 8 cellule totali. Blocca Fc-mediata anticorpo aspecifico legame utilizzando anti-CD16/CD32 anticorpi (0.5μg / 10 6 celle) per 30 minuti a 4 ° C. </li><li> Lavare le cellule con 10 ml di tampone di separazione a freddo e centrifugare per 10 minuti a 200 g a 4 ° C. Risospendere le cellule in 400 l di tampone di separazione. </li><li> Aggiungere 100 ml di microsfere CD11c al 10 8 </cellule sup&gt; totale. Mescolare bene e incubare per 15 minuti in frigorifero (2-8 ° C). </li><li> Lavare le cellule con 10 ml di tampone di separazione a freddo e centrifugare per 10 minuti a 200 g a 4 ° C e scartare il surnatante. </li><li> Risospendere il pellet cellulare in 3 ml di tampone di separazione a freddo. </li><li> Eseguire separazione magnetica utilizzando il autoMACS Pro separatore TM selezionando il posseld programma. Raccogliere la frazione positiva (0,5 ml). </li></ol> 3. Cellule dendritiche convenzionale (CDC) isolamento <ol><li> Incubare arricchito CD11c sospensione cellulare positivo con anti-CD11c PE-Cy7 e anti-IA/IE (MHC-II)-FITC anticorpi per 30 minuti a 4 ° C. Concentrazione ottimizzata di anticorpi è di 0,5 mcg / 10 6 delle cellule. </li><li> Lavare le cellule con il freddo BSA PBS/0.5% e centrifugare per 10 minuti a 335 x g a 4 ° C. Risospendere le cellule in 0,5 ml di PBS/0.5% BSA e passarli attraverso una maglia di nylon 40 micron. </li><li> Procedere allaordinare le celle utilizzando il FACS sorter Aria cellulare in modalità purezza. Aggiungere 100 ml di PBS/0.5% BSA ai tubi di raccolta al fine di prevenire i danni delle cellule purificate. Tenere le cellule ad una temperatura refrigerata per tutto il processo di separazione delle cellule. </li></ol> 4. Protocollo alternativo per ottenere una sospensione singola cellula e CD11c + arricchimento delle cellule <ol><li> In sostituzione del dissociatore gentleMACS, tessuto polmonare a terra a partire dal punto 1.5 può essere trasferito ad un tubo da 15 ml contenente 5 ml di soluzione di collagenasi al polmone e incubare per 1 ora a 37 ° C. Vortex cellule ogni 15 minuti per risospendere frammenti di tessuto. A distruggere il tessuto passando 6-8 volte il campione dentro e fuori attraverso una siringa da 3 ml, collegato ad un ago 20 G. Evitare di fare bolle durante il processo, altrimenti la vitalità cellulare sarà compromessa. Una volta sospensione singola cellula si ottiene, passare alla fase 1.8. </li><li> In sostituzione del TM AutoMACSPro separazionetor, l&#39;isolamento magnetico per l&#39;arricchimento delle cellule CD11c, può essere eseguita anche facendo passare la sospensione cellulare manualmente attraverso due colonne MACS. La selezione delle colonne appropriato varia a seconda della dimensione del campione. </li></ol> 5. Rappresentante dei risultati: Polmone CDC sono identificati come CD11c hi / MHC-II popolazione cellulare hi. Come mostrato in fig. 1, CDC rappresentano una percentuale minore del 1,4% rispetto ad alcuni altri tessuti come milza (4,8%). Tuttavia, a differenza di milza, polmone cellule CD11c positivo esprimere diverse quantità di MHC-II, tra cui una popolazione di cellule diverso da quello di CDC. Dopo la cella singola preparazione, la resa di cellule polmonari totale era di circa 3,0-3,8 x 10 7 cellule / polmonare con una vitalità del 60-70%, da cui poco più dell&#39;1% rappresentato sottoinsieme CDC. Questa percentuale è aumentata dopo l&#39;isolamento CD11c magnetico in cui è stato incrementato il totale del polmone CDC circa 10 tIME (&gt; 16%) rispetto al preparato originale (Fig. 2A). Tuttavia, le cellule che esprimono CD11c basse quantità di MHC-II (CD11c hi / lo MHC-II, macrofagi) ha rappresentato una popolazione di cellule ad alta contaminazione (&gt; 70%) che è stato mescolato con il sottoinsieme polmone CDC. Come mostrato in fig. 2B, queste cellule sono stati eliminati dopo la fase di separazione delle cellule quando la purezza CDC ha raggiunto&gt; 96%. Il rendimento abituale di CDC, dopo l&#39;intera procedura è stata di 5 x 10 4 cellule / polmone. Microfotografie mostrare caratteristiche morfologiche di CDC come cellule arrotondate con i dendriti tipico (pannello superiore). D&#39;altra parte, CD11c hi / MHC-II lo ha rappresentato un sottoinsieme morfologicamente differente che mostra protuberanze cellulari che sono tipici di macrofagi (MΦ, pannello inferiore) 12. <img alt="Figura 1" src="/files/ftp_upload/3563/3563fig1.jpg" /> Figura 1. Differenziale frequenza DC nel polmone del mouse e della milza. Polmone unomilza tessuti da C57BL ° / 6 topi sono stati raccolti e trattati con collagenasi. A seguito di digestione collagenasi, le cellule sono state colorate con anticorpi anti-mouse CD11c-PE-Cy7 e anti-topo IA / IE-FITC. Rappresentante trame citometria a flusso mostrano percentuali di CDC (CD11c hi / MHC-II hi) nei polmoni e nella milza. <img alt="Figura 2" src="/files/ftp_upload/3563/3563fig2.jpg" /> Figura 2. Isolamento di CDC dal polmone del mouse. Polmone del mouse sospensione cellulare è stato etichettato con anti-CD11c microsfere e passato attraverso un separatore cellulare automatico. A) Un grafico rappresentante mostra le percentuali di CDC (CD11c hi / MHC-II hi) e macrofagi (MΦ, CD11c hi / MHC-II lo) le popolazioni dopo arricchimento CD11c. Ulteriori colorazione della frazione arricchita seguita con anti-topo CD11c-PE-Cy7 e anti-topo IA / IE-FITC. Doppia cellule positive erano ordinati e analizzati mediante citometria a flusso. Aidentificare i loro morfologia, le cellule sono state cytospun e tinto con un Wright-Giemsa colorazione modificato. B) Rappresentante trame mostrano percentuali di CDC e MΦ dopo l&#39;ordinamento delle cellule. Microfotografie mostrare una immagine rappresentativa del polmone CDC e MΦ polmone. Barra di scala = 20 micron.

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Isolamento dei polmonare topo DC è una tecnica importante per lo studio di una vasta gamma di stimoli respiratori. Il processo per ottenere queste cellule include passaggi critici che impediscono la perdita di cellule così come la vitalità cellulare e la purezza. Perfusione del polmone prima della raccolta contribuirà a eliminare eventuali cellule periferiche e ridurre eritrociti contaminante. L&#39;utilizzo della dissociazione automatico può essere advanteogus quando un gran numero di polmoni vengono gestiti, altr...

<table width="200" border="1"><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Lisi buffer di ACK </td><td> Invitrogen </td><td> D6-0005DG </td><td></td></tr><tr><td> Anti-topo CD11c (HL3) </td><td> BD Pharmingen </td><td> 5580979 </td><td> PE-Cy7 coniugato </td></tr><tr><td> Anti-topo IA / IE (269) </td><td> BD-Pharmingen </td><td> 553623 </td><td> Coniugato con FITC </td></tr><tr><td> Collangenase tipo 1A </td><td> Sigma </td><td> 9891-500mg </td><td></td></tr><tr><td> Cellula filtri </td><td> BD Falcon </td><td> 352340, 352360 </td><td></td></tr><tr><td> CD11c (N418) microsfere </td><td> Miltenyi </td><td> 130-052-001 </td><td></td></tr><tr><td> DNasi I </td><td> Sigma </td><td> D5025-150KU </td><td></td></tr><tr><td> Sale di Hank soluzione equilibrata </td><td> Invitrogen </td><td> 14170 </td><td></td></tr><tr><td> Hepes soluzione tampone </td><td> Invitrogen </td><td> 15630 </td><td></td></tr><tr><td> Piastre di Petri 60 millimetri </td><td> BD Falcon </td><td> 351016 </td><td></td></tr><tr><td> GentleMACS ™ C tubi </td><td> Miltenyi </td><td> 130-093-237 </td><td></td></tr><tr><td> Gentle MACS dissociatore </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

Le cellule dendritiche del polmone (DC) svolgono un ruolo fondamentale nel rilevamento invadendo 1,2 patogeni così come nel controllo della risposta tollerogeniche 3 nel tratto respiratorio. Almeno tre principali sottoinsiemi di cellule dendritiche del polmone sono stati descritti nei topi: convenzionale DC (CDC) 4, plasmacitoidi DC (PDC) 5 e la produzione di IFN-DC assassino (IKDC) 6,7. Il sottoinsieme CDC è il sottoinsieme DC più importanti nel polmone 8. Il marcatore comune noto per identificare sottoinsiemi DC è CD11c, un tipo mi transmembrana integrina (β2), che si esprime anche sui monociti, macrofagi, neutrofili e alcune cellule B 9. In alcuni tessuti, utilizzando CD11c come marcatore per identificare il mouse DC è valido, come nella milza, dove la maggior parte delle cellule + CD11c rappresentano il sottoinsieme CDC che esprime alti livelli del complesso maggiore di istocompatibilità di classe II (MHC-II). Tuttavia, il polmone è un tessuto più eterogeneo dove esseresottoinsiemi lato DC, vi è un&#39;alta percentuale di una popolazione di cellule distinte che esprime alti livelli di CD11c attacco bassi livelli di MHC-II. Sulla base della sua caratterizzazione e soprattutto sulla sua espressione di F4/80, un marcatore dei macrofagi della milza, il CD11c hi-MHC II popolazione di cellule del polmone lo è stato identificato come i macrofagi polmonari 10 e più recentemente, come un potenziale precursore DC 11. In contrasto con il mouse pDC, lo studio del ruolo specifico del CDC nella risposta immunitaria polmonare è stata limitata a causa della mancanza di un marcatore specifico che potrebbe aiutare a l&#39;isolamento di queste cellule. Pertanto, in questo lavoro, si descrive una procedura per isolare altamente purificato del mouse polmone CDC. L&#39;isolamento di polmonare sottoinsiemi DC rappresenta uno strumento molto utile per acquisire conoscenze in funzione di queste cellule in risposta a patogeni respiratori così come i fattori ambientali che possono innescare la risposta immunitaria nei polmoni.

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

Isolation of Mouse Lung Dendritic Cells

Una preparazione altamente purificato di cellule dendritiche del polmone del mouse è descritto. Particolare enfasi è data l&#39;isolamento delle tradizionali sottoinsieme delle cellule dendritiche.

Isolamento di cellule polmonari mouse dendritiche

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 Views.  Louisiana State University. Una preparazione altamente purificato di cellule dendritiche del polmone del mouse è descritto. Particolare enfasi è data l'isolamento delle tradizionali sottoinsieme delle cellule dendritiche.

Video: 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 di cellule della cavità peritoneale mouse

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

Ricerca

Immunologia e infezioni

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 caratterizzazione di cellule dendritiche ei macrofagi dall&#39;intestino mouse

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.

Isolamento di cellule dendritiche mieloidi e cellule epiteliali di timo umano

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.

Un metodo del rendimento efficiente e di alta per l&#39;isolamento di mouse dendritiche sottopopolazioni cellulari

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.

Separazione delle cellule fluorescenza-attivato per la purificazione delle cellule dendritiche plasmacitoidi dal midollo osseo di topo

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.

Generazione di derivate da monociti cellule dendritiche umane da sangue intero

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 membrana esterna Vescicole: Isolamento e analisi del loro potenziale pro-infiammatori sui macrofagi

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.

Cellule umane del polmone dendritiche: distribuzione spaziale e fenotipica di identificazione in endobronchiale biopsie utilizzando l&#39;immunoistochimica e citometria a flusso

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.

Taglio di precisione polmone mouse Fette per la visualizzazione in diretta cellule dendritiche polmonare

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 di cellule dendritiche dal tratto riproduttivo femminile umano per studi fenotipici e funzionali

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