Diese Arbeit wurde von CIHR und CF Kanada Stipendien an Dr. Jim Hu und durch ein Doktoranden-Stipendium vergeben, um Rahul Kushwah von CF Kanada finanziert.

1. Sensibilisierung von Mäusen mit Ovalbumin <ol><li> Bereiten Sie eine Lösung von OVA (Grad V, Sigma, MO) in sterilem PBS in einer Konzentration von 1 mg / ml (Lösung kann bei -80 ° C gelagert werden). </li><li> Um OVA-Alaun Mischung herzustellen, um Alaun in einem Gefäß und fügen OVA-Lösung in einem tropfenweise unter Vortexen des Rohrs bei einem Verhältnis von 1:1. Rühren Sie die Mischung für 30 Minuten und sofort nutzen nach dem Mischen. </li><li> Verwendung einer 1 ml Spritze, Injektion 0,2 ml der Mischung in Maus-Peritonealhöhle und wiederholen Sie die Einspritzung wieder nach 2 Wochen. </li></ol> 2. Intranasalen Challenge of Mäuse mit FITC markiert Ovalbumin <ol><li> 7 Tage nach der zweiten Injektion von OVA-Alaun sind Mäuse abgebildet intranasal mit OVA-FITC in Frage gestellt werden. </li><li> Eine Lösung aus OVA-FITC (Sigma) unter Verwendung steriler PBS in einer Konzentration von 1 mg / ml aliquotiert und bei -80 ° C </li><li> Mit einem sterilen Gaze an der Unterseite eines 50 ml Falcon-Röhrchen und in einem chemischen hOOD, 5 ml Isofluran auf die Gaze. Um Mäuse betäuben, richten Sie das Tier in die Röhre für etwa 5-10 Sekunden. </li><li> Halten Sie den narkotisierten Tier in aufrechter Position und mit sterilen Pipettenspitzen, Pipette 100 ul der OVA-FITC-Lösung auf der Nase von Mäusen in einer tropfenweise Mode. </li><li> Wiederholen Sie die intranasale Provokation mit OVA-FITC für die nächsten zwei Tage. </li></ol> 3. Erstellung von Einzel-Zell-Suspension aus der Lunge und den Lymphknoten <ol><li> Opfere die Mäuse mit intraperitonealen Injektion von Euthanyl. </li><li> Um Makrophagen-Kontamination, die DC-Analyse erschweren können zu reduzieren, wird Lungenlavage durchgeführt. Um die Lungen zu spülen, werden Maus Luftröhren kurz belichtet und mit einem Katheter und einer Spritze durchbohrt; mit eiskaltem PBS mit 5 mM EDTA, der unteren Atemwege wird 3-mal gespült, um Zellen aus den alveolären Bereiche zu entfernen. </li><li> Versorgen die Lungen mit 10 ml PBS, enthaltend 10 U / ml Hepardie über den rechten Ventrikel des Herzens, um Blutzellen aus der Lungengefäße zu entfernen. Führen Sie die Infusionen, bis die Lungen weiß, was auf Entfernung des größten Teils der Blutzellen verwandeln. Dies ist besonders wichtig, um kontaminierende Zellen aus dem peripheren Blut zu entfernen. </li><li> Sezieren die Lunge heraus und entfernen Sie mediastinalen Lymphknoten (MLN) und platzieren Sie die Lymphknoten in einer separaten Schüssel. (Versuchen Sie, um die Exposition der Gewebe zum Vorschein zu Quenching der Fluoreszenz zu verhindern, zu minimieren.) </li><li> Legen Sie die Lungen auf einer Petrischale und Hackfleisch mit einer Schere. Nächste verdauen die Lunge für 25 Minuten bei 37 ° C unter Verwendung von 250 U / ml Kollagenase D (Roche) und fügen EDTA (10 mM Endkonzentration) für die nächsten 5 Minuten, um Kollagenase-Aktivität zu stoppen. </li><li> Führen Sie die Fragmente verdaut Lunge durch ein 100 um Sieb Zelle und führen hypotone Lyse an Erythrozyten zu entfernen. Die einzigen Zellsuspension ist bereit für die weitere Analyse der DCs. </li><li> Um einzelne Zellsuspension aus der MLN, t vorbereitenerleichtern den MLN mit feinen Nadeln und Verdau mit Kollagenase wie oben beschrieben, gefolgt von Pressen durch ein Sieb Zelle. </li></ol> 4. Die Färbung für DC-Marker zur Reifung / Migration Beurteilen <ol><li> Um DCs in der Lunge zu erkennen, ist Färbung für CD11c und CD11b durchgeführt. Außerdem, für Reifung zu beurteilen, ist Färbung für CD86 und CD80, die hochreguliert als DCs Reifung unterzogen werden durchgeführt. CD11c + CD11b + OVA-FITC +-Zellen in der MLN werden als pulmonale DCs die Migration von der Lunge zu den MLN als Antwort auf OVA Atemwege Herausforderung identifiziert. </li><li> Suspend-Zellen in einer Konzentration von 10 × 10 6 Zellen / ml in FACS-Puffer (PBS mit 1% FBS und 1 mM EDTA) und Aliquot 150 ul Zellsuspension / Röhre für FACS-Färbung. Zur Identifizierung von Lungen-DCs, nach Flecken werden benötigt: Ungefärbte Kontrolle (Lung Zellen von Mäusen nicht zu OVA-FITC ausgesetzt), OVA-FITC-Kontrolle, CD11c einzige Kontrolle, CD11b einzige Kontrolle, CD11b+ CD11c + Doppel-Kontrolle, CD11b + OVA-FITC + doppelte Kontrolle, CD11c + OVA-FITC + doppelte Kontrolle, MHC II einzigen Kontrolle, CD86 einzige Kontrolle, CD80 einzigen Kontrolle und Proben für die OVA-FITC + CD11c + CD11b + + MHCII gefärbt , OVA-FITC + CD11c + CD11b + CD86 + und OVA-FITC + CD11c + CD11b + CD80 +. </li><li> Um DC-Migration zu bewerten, führen absolute Zellzahlen der einzige Zellsuspension aus MLN und fertigt sodann folgende Röhren: Ungefärbte Kontrolle (MLN-Zellen von Mäusen, die nicht auf OVA-FITC ausgesetzt), OVA-FITC-Kontrolle, CD11c + einzigen Kontrolle, CD11b + doppelte Kontrolle, CD11b + OVA-FITC + Doppel-Kontrolle, CD11c + OVA-FITC + Doppel-Steuerung und CD11c + CD11b + OVA-FITC + Proben. </li></ol> 5. Repräsentative Ergebnisse Die Zeitpunkte für die intraperitoneale Sensibilisierung erforderlich ist, um Atemwege allergische Entzündung zu induzieren ist wichtig und sollte durchgeführt werden, wie in 1 dargestellt. Nach intraperitonealer Sensibilisierung und Atemwege OVA Herausforderung, die Induktion von allergischen Entzündung der Atemwege zu bestätigen, können einige Mäuse getötet und histologische Analysen auf der Lunge Abschnitten durchgeführt werden, wie in 2 gezeigt. Vorliegen von Entzündungszellen kann durch Hämatoxylin &amp; Eosin-Färbung bestätigt werden (Abbildung 2A) und Anwesenheit von Schleim Produktion kann durch periodische-Acid-Schiff-Färbung (Abbildung 2B) beurteilt werden. Insgesamt stellt die Induktion der allergischen Entzündung der Atemwege nach OVA Herausforderung der OVA-sensibilisierten Mäusen 8 zu bestätigen. Im Gegensatz dazu werden Lungenschnitten von Saline behandelten Mäusen zu erwarten, dass frei von Entzündungen mit Fehlen einer Schleimbildung. Darüber hinaus folgende OVA Herausforderung, Lungen-DCs unde<html> rgo Reifung und spätere Migration zu den Lymphknoten. Analyse der CD11c + Zellen in den Lymphknoten (MLN) aus sensibilisierten Mäusen mit OVA und herausgefordert wird erwartet, dass ein höherer Anteil der CD11c + Zellen im Vergleich zu Kochsalzlösung behandelten Mäusen (3A) zu zeigen. Außerdem Analyse der absoluten Zellzahl von CD11c + CD11b + OVA-FITC +-Zellen in der MLN von OVA-sensibilisierten und provozierten Mäusen, wird erwartet, zeigen eine signifikant höhere (dh um ein Mehrfaches höher) zählen, dass die Kollegen aus Kochsalzlösung behandelten Mäusen ( 3B). <img alt="1" src="/files/ftp_upload/4014/4014fig1.jpg" /> 1. Experimentelles Protokoll zur Induktion von Ovalbumin (OVA) induzierten allergischen Entzündung der Atemwege bei Mäusen mit Atemwege Herausforderung mit FITC-markiertem Ovalbumin (OVA-FITC). fig2.jpg &quot;/&gt; 2. OVA Sensibilisierung durch OVA Atemwege Herausforderung gefolgt führt zur Induktion von allergischen Entzündung der Atemwege, wie identifiziert durch Hämatoxylin und Eosin-Färbung der Lunge Abschnitte (A) gezeigt, und führt auch zu Schleimproduktion in den Atemwegen, wie durch Periodsäure-Schiff-Färbung der Lunge festgestellt Abschnitte, wie in (B) gezeigt. <img alt="Abbildung 3" src="/files/ftp_upload/4014/4014fig3.jpg" /> Abbildung 3. OVA-FITC Herausforderung induziert DC-Migration von der Lunge zu den mediastinalen Lymphknoten (MLN). (A) Durchflusszytometrie Plots darstellen Anteile von CD11c + Zellen in der Kontrolle oder der MLN OVA-sensibilisierten Mäusen. (B) Absolute Grafen von CD11c + CD11b + OVA-FITC +-Zellen in der MLN auf Salz-oder OVA-sensibilisierten Mäusen. * P &lt;0,05. <a href="http://www.jove.com/files/ftp_upload/4014/4014fig3large.jpg" target="_blank">Klicken Sie hier für eine größere Abbildung anzuzeigen</a> .

<ol>
	<li>Kushwah, R., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+dendritic+cells+in+the+induction+of+regulatory+T+cells.">Role of dendritic cells in the induction of regulatory T cells.</a> Cell Biosci. 20, (2011).</li><li>Kushwah, R., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexity+of+dendritic+cell+subsets+and+their+function+in+the+host+immune+system.">Complexity of dendritic cell subsets and their function in the host immune system.</a> Immunology. 133, 409-419 (2011).</li><li>Lambrecht, B. N., Hammad, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+our+breath+away:+dendritic+cells+in+the+pathogenesis+of+asthma.">Taking our breath away: dendritic cells in the pathogenesis of asthma.</a> Nat Rev Immunol. 3, 994-1003 (2003).</li><li>Hammad, H., Lambrecht, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+epithelial+cells:+linking+innate+and+adaptive+immunity+in+asthma.">Dendritic cells and epithelial cells: linking innate and adaptive immunity in asthma.</a> Nat. Rev. Immunol. 8, 193-204 (2008).</li><li>Lloyd, C. M., Murdoch, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerizing+allergic+responses+in+the+lung.">Tolerizing allergic responses in the lung.</a> Mucosal. Immunol. 3, 334-344 (2010).</li><li>Herz, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+of+murine+animal+models+to+study+the+development+of+allergic+bronchial+asthma.">The relevance of murine animal models to study the development of allergic bronchial asthma.</a> Immunol. Cell Biol. 74, 209-217 (1038).</li><li>Herz, U., Lumpp, U., Daser, A., Gelfand, E. W., Renz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+animal+models+to+study+the+central+role+of+T+cells+in+immediate-type+hypersensitivity+responses.">Murine animal models to study the central role of T cells in immediate-type hypersensitivity responses.</a> Adv. Exp. Med. Biol. 409, 25-32 (1996).</li><li>Nakae, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF+can+contribute+to+multiple+features+of+ovalbumin-induced+allergic+inflammation+of+the+airways+in+mice.">TNF can contribute to multiple features of ovalbumin-induced allergic inflammation of the airways in mice.</a> J. Allergy Clin. Immunol. 119, 680-686 (2007).</li><li>Guth, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+environment+determines+unique+phenotype+of+alveolar+macrophages.">Lung environment determines unique phenotype of alveolar macrophages.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 296, L936-L946 (2009).</li><li>Kushwah, R., Cao, H., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+pulmonary+T+cell+response+to+helper-dependent+adenoviral+vectors+following+intranasal+delivery.">Characterization of pulmonary T cell response to helper-dependent adenoviral vectors following intranasal delivery.</a> J. Immunol. 180, 4098-4108 (2008).</li><li>Kushwah, R., Oliver, J. R., Zhang, J., Siminovitch, K. A., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+dendritic+cells+induce+tolerance+in+mice+through+suppression+of+dendritic+cell+maturation+and+induction+of+antigen-specific+regulatory+T+cells.">Apoptotic dendritic cells induce tolerance in mice through suppression of dendritic cell maturation and induction of antigen-specific regulatory T cells.</a> J. Immunol. 183, 7104-7118 (2009).</li></ol>

Keine Interessenskonflikte erklärt.

Die hier vorgestellte Methode bietet eine Durchflusszytometrie basierenden Ansatz für die Analyse von Lungen-DCs, bei Lieferung von OVA-FITC zur Sicherung der Atemwege Herausforderung basiert. Dies ermöglicht eine selektive Abfrage der pulmonalen DCs, die bis zu OVA-FITC und damit die DC-Populationen, die überwacht werden, sind diejenigen, die effektiv in den Atemwegen Immunantwort beteiligt sind im Verlauf der OVA-induzierten allergischen Entzündung der Atemwege. In Kontroll-Mäusen, in Abwesenheit von allergischen...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Ovalbumin </td><td> Sigma-Aldrich </td><td> A5503 </td></tr><tr><td> Alaun </td><td> Thermo Scientific </td><td> 77161 </td></tr><tr><td> OVA-FITC </td><td> Sigma-Aldrich </td><td> A9771 </td></tr><tr><td> Collagenase D </td><td> Roche </td><td> 11088858001 </td></tr><tr><td> Antikörper </td><td> E-Bioscience </td><td></td></tr></tbody></table>

analysis of pulmonary dendritic cell maturation and migration during allergic airway inflammation

Dendritische Zellen (DCs) sind die Hauptakteure bei der Initiation der adaptiven Immunantwort durch Aktivierung Antigen-spezifischen T-Zellen beteiligt. DCs vorhanden sind, in den peripheren Geweben im eingeschwungenen Zustand, aber als Reaktion auf Antigen-Stimulation, nehmen DCs das Antigen und wandern schnell zu den Lymphknoten, wo sie initiieren T-Zell-Antwort gegen das Antigen 1,2. Zusätzlich DCs spielen auch eine entscheidende Rolle bei der Initiierung Autoimmun sowie allergische Immunantwort 3. DCs spielen eine wesentliche Rolle sowohl in der Initiierung der Immunantwort und die Induktion einer Toleranz in der Einstellung der Lunge Umwelt 4. Lung Umgebung weitgehend tolerogene aufgrund der Exposition gegenüber Vielzahl von Umweltantigene 5. Doch bei einigen Personen gibt es eine Pause in der Toleranz, die zur Induktion von Allergien und Asthma führt. In dieser Studie beschreiben wir eine Strategie, die zur Reifung und Atemwege DC MIG überwacht werden könnenRation in Reaktion auf das Antigen zur Sensibilisierung verwendet. Die Messung der Atemwege Reifung und Migration ermöglicht die Beurteilung der Kinetik der Immunantwort bei allergischen Entzündung der Atemwege und hilft beim Verständnis der Größe der nachfolgende Immunreaktion zusammen mit den zugrunde liegenden Mechanismen. Unsere Strategie basiert auf der Verwendung von Ovalbumin als Sensibilisierungsmittel bezogen. Ovalbumin-allergischen Asthma ist eine weit verbreitete Modell, um die Lungeneosinophilie, Lungenentzündung und erhöhte IgE-Spiegel bei Asthma 6,7 vor reproduzieren. Nach Sensibilisierung werden die Mäuse durch intranasale Verabreichung von FITC-markierten Ovalbumin, die zur spezifischen Markierung von Atemwegs-DCs, welche die Aufnahme ermöglicht Ovalbumin herausgefordert. Als nächstes mit mehreren DC-spezifische Marker, können wir beurteilen, die Reifung der DC und kann auch beurteilen, ihre Wanderung zu den Lymphknoten durch den Einsatz der Durchflusszytometrie.

Watch this Scientific Journal Video about Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation at JoVE.com

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Wir beschreiben eine Strategie zur Reifung und Migration der pulmonalen dendritischen Zellen als Reaktion auf Ovalbumin in der Einstellung von Eieralbumin allergischen Entzündung der Atemwege zu überwachen. Diese Strategie kann modifiziert werden, um die Migration der pulmonalen dendritischen Zellen in den Einstellungen der Infektion herangezogen werden.

Analyse der Lungen-Reifung von dendritischen Zellen und Migration während der allergischen Atemwegsentzündung

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

analysis-pulmonary-dendritic-cell-maturation-migration-during

Research

JoVE Journal

Immunology and Infection

14.9K Aufrufe.  McMaster University, Hamilton. Wir beschreiben eine Strategie zur Reifung und Migration der pulmonalen dendritischen Zellen als Reaktion auf Ovalbumin in der Einstellung von Eieralbumin allergischen Entzündung der Atemwege zu überwachen. Diese Strategie kann modifiziert werden, um die Migration der pulmonalen dendritischen Zellen in den Einstellungen der Infektion herangezogen werden.

Serie: Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Menschliche T-Lymphozyten-Isolierung, Kultur und Analyse der Migration In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Forschung

Immunologie und Infektion

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Optimiertes Protokoll für die effiziente Transfektion von dendritischen Zellen ohne Zell-Reifung

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Durchflusszytometrie-Analyse von Immunzellen innerhalb Murine Aorten

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Überwachung dendritische Zellen mit Migration<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H Magnetic Resonance Imaging

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

In-vivo-Imaging-Methode zu unterscheiden, akuten und chronischen Entzündungen

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Isolierung, Charakterisierung und funktionelle Untersuchung des Zahnfleisch Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

Intratracheale Verabreichung von<em&gt; Haemophilus influenzae</em&gt; In Mausmodelle zur Untersuchung Atemwegsentzündung

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

Ein CFSE-basierten Assay die Migration von murinen Haut Dendritischen Zellen in Lymphknoten während der Infektion zu studieren mit<em&gt; Mycobacterium bovis</em&gt; Bazillus Calmette-Guérin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

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 ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Analyse der Scherung strömungsinduzierte Migration der Murine Marginal Zone B-Zellen In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...