Questo lavoro è stato finanziato dalla CIHR e CF Canada concede al dottor Jim Hu e da una borsa di studio di dottorato assegnato a Rahul Kushwah da CF Canada.

1. Sensibilizzazione dei topi con ovoalbumina <ol><li> Preparare una soluzione di OVA (grado V, Sigma, MO) in PBS sterile a una concentrazione di 1 mg / ml (soluzione può essere conservato a -80 ° C). </li><li> Al fine di preparare OVA-Alum miscela, prendere allume in un tubo e aggiungere la soluzione di OVA in maniera a gocce mentre il tubo vortex ad un rapporto di 1:1. Mescolare il composto per 30 minuti e usare subito dopo la miscelazione. </li><li> Uso di una siringa da 1 ml, iniettare 0,2 ml della miscela nella cavità peritoneale del mouse e ripetere l&#39;iniezione dopo 2 settimane. </li></ol> 2. Sfida intranasale di topi con FITC ovoalbumina etichetta <ol><li> 7 giorni dopo la seconda iniezione di OVA-Alum, i topi sono pronti per essere inoculati per via intranasale con OVA-FITC. </li><li> Preparare una soluzione di OVA-FITC (Sigma) usando PBS sterile a una concentrazione di 1 mg / ml e conservare in aliquote a -80 ° C. </li><li> Inserire una garza sterile sul fondo di un tubo Falcon da 50 ml e in uno h chimicoood, aggiungere 5 ml di Aerrane sulla garza. Allo scopo di anestetizzare topi, dirigere l&#39;animale nel tubo per circa 5-10 secondi. </li><li> Tenere l&#39;animale anestetizzato in posizione verticale e l&#39;utilizzo di puntali sterili, Pipettare 100 ul del OVA-FITC soluzione sulle narici di topi in una goccia a goccia moda. </li><li> Ripetere sfida intranasale con OVA-FITC per i prossimi due giorni. </li></ol> 3. Preparazione di singole cellule Sospensione dei polmoni e dei linfonodi che drenano <ol><li> Sacrifica i topi con l&#39;iniezione intraperitoneale di Euthanyl. </li><li> Al fine di ridurre la contaminazione dei macrofagi, che può complicare l&#39;analisi DC, lavaggio polmonare viene eseguita. Per lavanda i polmoni, trachee mouse sono brevemente esposto e cannulata con un catetere e una siringa, utilizzando PBS ghiacciato di 5 mM EDTA, il tratto respiratorio inferiore è risciacquato 3 volte per rimuovere le cellule dagli spazi alveolari. </li><li> Perfondere i polmoni con 10 ml di PBS contenente 10 U / ml heparin attraverso il ventricolo destro del cuore, per rimuovere le cellule del sangue dal sistema vascolare polmonare. Eseguite le perfusioni fino ai polmoni diventare bianchi, indicativo di rimozione della maggior parte delle cellule del sangue. Questo è particolarmente importante rimuovere cellule contaminanti dal sangue periferico. </li><li> Staccare i polmoni e rimuovere i linfonodi mediastinici (MLN) e porre i linfonodi in un piatto a parte. (Provare a minimizzare l&#39;esposizione dei tessuti alla luce per evitare lo spegnimento del FITC.) </li><li> Posizionare i polmoni su una piastra di Petri e tritare con le forbici. Avanti digerire i polmoni per 25 minuti a 37 ° C con 250 U / ml Collagenasi D (Roche) e aggiungere EDTA (10 mM di concentrazione finale) per i prossimi 5 minuti per fermare le attività collagenasi. </li><li> Passare i frammenti dei polmoni digeriti attraverso un colino 100 pm cellulare e procedere alla lisi ipotonica per rimuovere eritrociti. La sospensione singola cellula è pronto per ulteriori analisi delle DC. </li><li> Per preparare sospensione di cellule singole dal MLN, tfacilitare la MLN utilizzando aghi sottili e digerire con collagenasi come descritto in precedenza seguita da sforzo attraverso un colino cella. </li></ol> 4. La colorazione per DC Marcatori per valutare Maturazione / migrazione <ol><li> Al fine di identificare DC nei polmoni, colorazione viene eseguita per CD11c e CD11b. Inoltre, per valutare per la maturazione, la colorazione viene eseguita per CD86 e CD80, che sono upregulated come controller di dominio sottoposti a maturazione. CD11c + CD11b + OVA-FITC le cellule + nel MLN sono identificati come DCs polmonari migratori dai polmoni al MLN in risposta alla sfida delle vie aeree OVA. </li><li> Sospensione cellule ad una concentrazione di 10 x 10 6 cellule / ml in tampone FACS (PBS con 1% FBS e 1 mM EDTA) e un&#39;aliquota 150 microlitri di sospensione cellulare / provetta per colorazione FACS. Per l&#39;identificazione dei PVS polmonari, le macchie sono necessari: avviare l&#39;elaborazione di controllo (cellule polmonari di topi non esposti a OVA-FITC), OVA-FITC, controllo CD11c singolo, CD11b singolo controllo, CD11bCD11c + + doppio controllo, CD11b + OVA-FITC + doppio controllo, CD11c + OVA-FITC + doppio comando, MHC II singolo controllo, controllo CD86 singolo controllo CD80 singolo e campioni colorati per OVA-FITC + CD11c + CD11b + + MHCII , OVA-FITC + CD11c + CD11b + CD86 + e OVA-FITC + CD11c + CD11b + CD80 +. </li><li> Per valutare la migrazione DC, eseguire la conta delle cellule assoluti della sospensione cellulare singolo MLN e successivamente preparare i tubi seguenti: controllo avviare l&#39;elaborazione MLN (cellule di topi non esposti a OVA-FITC), OVA-FITC controllo, CD11c + di controllo unico, CD11b + doppio controllo, CD11b + OVA-FITC + doppio comando, CD11c + OVA-FITC + doppio controllo e CD11c + CD11b + OVA-FITC + campioni. </li></ol> 5. Risultati rappresentativi I punti di tempo necessari per la sensibilizzazione intraperitoneale per indurre infiammazione allergica delle vie aeree è importante e deve essere eseguita come illustrato in Figura 1. Dopo sensibilizzazione intraperitoneale e OVA sfida vie aeree, per confermare l&#39;induzione delle vie aeree allergica, alcuni topi possono essere sacrificati e analisi istologica può essere eseguita su sezioni polmonari come mostrato nella Figura 2. La presenza di cellule infiammatorie può essere confermata da ematossilina e eosina (Figura 2A) e presenza di produzione di muco può essere valutata mediante periodica-acido-Schiff macchia (Figura 2B). In tutto questo conferma l&#39;induzione di infiammazione delle vie aeree allergica sfida OVA OVA di topi sensibilizzati 8. Al contrario, le sezioni polmonari da Saline topi trattati dovrebbero essere esenti da qualsiasi infiammazione lungo con assenza di qualsiasi produzione di muco. Inoltre, in seguito sfida OVA, polmonare DC unde<html> RGO maturazione e successiva migrazione verso i linfonodi drenanti. Analisi dei CD11c + cellule nei linfonodi mediastinici (MLN) da topi sensibilizzati e stimolati con OVA dovrebbe mostrare una maggiore percentuale di cellule CD11c + rispetto a topi trattati con soluzione salina (Figura 3A). Inoltre, l&#39;analisi della conta assoluta delle cellule del CD11c + CD11b + OVA-FITC + cellule in MLN di topi OVA sensibilizzate e contestato, dovrebbe mostrare una significativamente più alta (vale a dire diverse volte superiore) conta che le controparti di topi trattati con soluzione salina ( Figura 3B). <img alt="Figura 1" src="/files/ftp_upload/4014/4014fig1.jpg" /> Figura 1. Protocollo sperimentale per l&#39;induzione di ovoalbumina (OVA) indotta infiammazione delle vie respiratorie allergiche nei topi insieme con la sfida delle vie aeree con marcato con FITC ovoalbumina (OVA-FITC). fig2.jpg &quot;/&gt; Sensibilizzazione Figura 2. OVA seguita da sfida vie aeree OVA porta a induzione delle vie aeree allergica come identificato dalla colorazione con ematossilina e eosina di sezioni polmonari indicate in (A) e porta anche alla produzione di muco nelle vie aeree come identificati mediante colorazione di acido periodico Schiff polmone sezioni come illustrato in (B). <img alt="Figura 3" src="/files/ftp_upload/4014/4014fig3.jpg" /> Figura 3. OVA-FITC sfida induce la migrazione DC dai polmoni ai linfonodi mediastinici (MLN). (A) appezzamenti di citometria a flusso raffigurante proporzioni di CD11c + cellule in MLN di controllo o di OVA topi sensibilizzati. (B) conte assolute dei CD11c + CD11b + OVA-FITC celle + in MLN di topi saline o OVA-sensibilizzato. * P &lt;0,05. <a href="http://www.jove.com/files/ftp_upload/4014/4014fig3large.jpg" target="_blank">Clicca qui per ingrandire la figura</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>

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Il metodo qui presentato offre un approccio basato citometria a flusso per l&#39;analisi DC polmonari, sulla base di consegna di OVA-FITC per la sfida delle vie aeree. Questo consente il monitoraggio selettivo delle DC polmonari, che assorbono OVA-FITC e quindi le popolazioni DC, che sono monitorati sono effettivamente quelli che partecipano nella risposta immunitaria vie aeree durante il corso di OVA indotta infiammazione delle vie aeree allergica. In topi di controllo, in assenza di infiammazione delle vie aeree aller...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Ovalbumina </td><td> Sigma-Aldrich </td><td> A5503 </td></tr><tr><td> Allume </td><td> Thermo Scientific </td><td> 77161 </td></tr><tr><td> OVA-FITC </td><td> Sigma-Aldrich </td><td> A9771 </td></tr><tr><td> Collagenasi D </td><td> Roche </td><td> 11088858001 </td></tr><tr><td> Anticorpi </td><td> e-Bioscience </td><td></td></tr></tbody></table>

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

Le cellule dendritiche (DC) sono i principali attori coinvolti in avvio di risposta immunitaria adattativa mediante l&#39;attivazione antigene-specifiche cellule T. DC sono presenti nei tessuti periferici in stato stazionario, ma in risposta alla stimolazione antigene, DC prendere l&#39;antigene e migrare rapidamente i linfonodi drenanti dove iniziano risposta di cellule T contro il 1,2 antigene. Inoltre, DC svolgono un ruolo chiave nell&#39;attivare autoimmune nonché allergica risposta immunitaria 3. Dendritiche svolgono un ruolo essenziale nel sia all&#39;inizio della risposta immunitaria e l&#39;induzione di tolleranza nel contesto di ambienti polmone 4. Ambiente polmonare è largamente tollerogenica, a causa della esposizione vasta gamma di antigeni ambientali 5. Tuttavia, in alcuni individui vi è un&#39;interruzione in tolleranza, che porta ad induzione delle allergie e asma. In questo studio, si descrive una strategia, che può essere utilizzato per monitorare la maturazione delle vie aeree DC e migrazione in risposta per l&#39;antigene usato per la sensibilizzazione. La misurazione di maturazione delle vie aeree DC e migrazione permette di valutare la cinetica della risposta immunitaria durante infiammazione delle vie aeree allergica e aiuta anche a comprendere la grandezza della risposta immunitaria successivo insieme con i meccanismi sottostanti. La strategia si basa sull&#39;uso di ovalbumina come agente sensibilizzante. Ovalbumina indotta l&#39;asma allergico è un modello ampiamente utilizzato per riprodurre l&#39;eosinofilia delle vie aeree, l&#39;infiammazione polmonare ed elevati livelli di IgE trovati durante l&#39;asma 6,7. Dopo la sensibilizzazione, i topi sono sfidati con la consegna intranasale di ovoalbumina marcato con FITC, che permette di un&#39;etichettatura specifica dei PVS delle vie aeree che ovoalbumine assorbimento. Successivamente, utilizzando diversi marcatori specifici DC, possiamo valutare la maturazione di queste cellule dendritiche e può anche valutare la loro migrazione verso i linfonodi che drenano impiegando la citometria a flusso.

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

Descriviamo una strategia per monitorare la maturazione e la migrazione delle cellule dendritiche polmonari in risposta ovalbumina nel contesto di infiammazione delle vie aeree ovalbumina indotta allergica. Questa strategia può essere modificato per valutare la migrazione di cellule dendritiche in polmonari impostazioni di infezione.

Analisi della maturazione polmonare delle cellule dendritiche e migrazione durante l&#39;infiammazione allergica delle vie aeree

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

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JoVE Journal

Immunology and Infection

14.9K Views.  McMaster University, Hamilton. Descriviamo una strategia per monitorare la maturazione e la migrazione delle cellule dendritiche polmonari in risposta ovalbumina nel contesto di infiammazione delle vie aeree ovalbumina indotta allergica. Questa strategia può essere modificato per valutare la migrazione di cellule dendritiche in polmonari impostazioni di infezione.

Video: 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.

Isolamento linfociti T umani, la cultura e l&#39;analisi delle migrazioni 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 ...

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Immunologia e infezioni

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.

Protocollo ottimizzato per Transfection efficiente delle cellule dendritiche, senza la maturazione delle cellule

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.

Analisi Citometria a flusso di cellule immunitarie all&#39;interno aorte Murine

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.

Monitoraggio migrazione delle cellule dendritiche usando<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 metodo di imaging per distinguere infiammazione acuta e cronica

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.

Isolamento, caratterizzazione e esame funzionale della cella di rete gengivale immunitario

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.

Intra-tracheale Amministrazione<em&gt; Haemophilus influenzae</em&gt; In modelli murini per studiare infiammazione delle vie aeree

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.

Un test CFSE-based per studiare la migrazione delle cellule dendritiche murini della pelle in drenanti linfonodi durante l&#39;infezione con<em&gt; Mycobacterium bovis</em&gt; Bacille 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.

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 ...

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.

Analisi di taglio indotta da flusso di migrazione delle cellule di B di zona marginale murino 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, ...