Gli autori desiderano ringraziare il Dott. Alain Trautmann, che ci ha incoraggiato a compiere fette linfonodi. Questo lavoro è stato sostenuto in parte da finanziamenti della Ligue Nationale Contre le Cancer, la Fondation pour la Recherche Médicale en France e l&#39;Association pour la recherche sur le Cancer.

1. Preparare le fette di linfonodi <ol><li> Preparare un 4% a basso punto di fusione soluzione di agarosio per l&#39;incorporamento. Diluire agarosio in PBS e forno a microonde questa soluzione. Quando l&#39;agarosio è completamente sciolto, mantenere la soluzione a 37 ° C fino al momento dell&#39;utilizzo. </li><li> Preparare un 6-e piastra di coltura dei tessuti. Aggiungere 1,1 ml terreno di coltura RPMI completo per bene, e inserire un filtro organotipica in ciascun pozzetto. Posizionare il 6 pozzetti a 4 ° C fino al momento di trasferire fette. </li><li> Luogo rondelle in acciaio inossidabile, con diametro interno di 4 mm, in un piatto di plastica riempita con mezzo di RPMI completo. Rondelle saranno ulteriormente utilizzati per concentrare le cellule sulla fetta tagliata vibratome. </li><li> Sacrificare il mouse in conformità alla normativa vigente sul benessere degli animali. Rimuovere con cura i linfonodi periferici dal tessuto circostante e metterli in un piatto di plastica contenente ghiacciata PBS. Bisogna fare attenzione a non danneggiare la struttura dei linfonodi, in quanto questi organi sono molto morbide. </li><li> Versare il 4%-gel in una da 35 mm piatto di plastica e delicatamente trasferire i nodi nel gel. Lasciare il gel in ghiaccio per 5 minuti a indurire. </li><li> Rimuovere il blocco dal piatto e tagliare l&#39;agar in modo da lasciare 3-5 mm di gel intorno ad ogni linfonodo. </li><li> Collegare i nodi incorporato nel disco campione del vibratome con colla non tossica dei tessuti. Installare il disco campione nel vassoio pieno di ghiaccio PBS freddo. </li><li> Sezione l&#39;agar-embedded tessuto a 320 micron di spessore con la velocità vibratome impostato su una gamma lenta (0,3 mm / s) e la frequenza di vibrazione impostata su un medio raggio (1,5 mm). </li><li> Cura il trasferimento fette linfonodi mentre vengono tagliati sul inserti cultura organotipica utilizzando una pinza sottile. Posto 3 fette su ogni inserto. Fare molta attenzione come fette può essere facilmente danneggiato. Un nodo tipico linfonodi periferici produrrà 5 sezioni al taglio a 320 micron. Eliminare le fette prima e l&#39;ultima, in quanto contengono solo i tessuti superficiali. </li><li> Luogo rondelle in acciaio inossidabile su ogni fetta individuali. Assicurarsi che le rondelle sono ben posizionate sul agarosio che circonda il tessuto. </li><li> Incubare la piastra di coltura a 37 ° C in un 5% di CO 2 incubatore umidificato fino al momento di piatto cellule T. </li></ol> 2. Isolare le cellule T da linfonodi del mouse <ol><li> Isolare le cellule T ai sensi dell&#39;articolo precedentemente pubblicato JOVE 3. </li><li> Utilizzare le cellule T e passare immediatamente alla sezione successiva. In caso contrario, i linfociti possono essere colti durante la notte in completo RPMI-medium supplementato con 10 ng / ml di IL-7. </li></ol> 3. L&#39;etichettatura di isolate cellule T <ol><li> Lavare le cellule in HBSS. Risospendere loro nel buffer stesso a 10 x 10 6 per ml. </li><li> Aggiungere lo stesso volume di HBSS contenente 1 mM cellulare Tracker verde CMFDA dando una concentrazione finale di 0,5 micron. </li><li> Incubare la sospensione cellulare a 37 ° C per 5 min. Lavare le cellule due volte con RPMI completo e medie. </li><li> Risospendere le cellule ad una concentrazione finale di 10 x 10 6 per ml in completo RPMI-media. Queste cellule etichettate saranno placcato sulla fette. </li></ol> Altri coloranti fluorescenti che CMFDA potrebbe essere usato anche, ma di tenere presente che queste molecole sono potenzialmente tossici sopra determinate concentrazioni. 4. Placcatura cellule etichettati T su fette di linfonodi <ol><li> Rimuovere l&#39;eccesso di medie contenute nella parte interna della lavatrice con una pipetta. Non lasciare che il tessuto di diventare secco; lavorare velocemente durante questa fase. </li><li> Delicatamente dispensare 10-20 cellule T microlitri etichettati che corrisponde a 1 a 2 x 10 5 cellule della parte interna della lavatrice. Bisogna fare attenzione a non toccare il tessuto con la punta della pipetta. Assicurarsi che la goccia di sospensione cellulare rimane al suo posto. </li><li> Mettere le fette con le cellule in incubatore a 37 ° C e 5% di CO 2 per almeno 30 minuti per consentire la migrazione dei linfociti nel tessuto. </li></ol> 5. Imaging cellule T all&#39;interno fette linfonodi In questo video-articolo, le cellule T immagine fluorescente a fette linfonodale con un microscopio invertito widefield e un microscopio confocale in posizione verticale. <ol><li> Impostare la temperatura a 37 ° C. I nostri microscopi sono dotati di camere a temperatura controllata. </li><li> Installare un sistema di perfusione che permette perfusione continua della fetta linfonodale con ossigenato (5% CO 2, 95% O 2) rosso fenolo senza medium RPMI. Nel nostro sistema, perfusi dei media entra nella camera di immagini da una parte per gravità. Il mezzo viene aspirato con una pompa collegata ad un pallone di raccolta dei rifiuti. Impostare la portata media di 1 ml / min. </li><li> Con una pinza sottile, togliere delicatamente la fetta da 6 pozzetti e immergere la preparazione in riscaldato medio ossigenato RPMI per qualche secondo per liberarsi di cellule fluorescenti non infiltrato nel tessuto. </li></ol> ImaGing cellule T con un ampio campo di microscopio invertito <ol start="4"><li> Mettere la fetta a testa in giù su una misura da camera di imaging che consiste in fili di nylon incollato sulla parte interna di un piatto fondo di vetro. In questa camera, i fili di nylon elevare la fetta dal vetro e consentire la soluzione ossigenata di diffondere nel tessuto. Ciò è necessario come la migrazione delle cellule T nei linfonodi è fortemente dipendente da ossigeno. Fissare la fetta su di esso con l&#39;aggiunta di una rondella in acciaio inox. In queste condizioni, la fetta si trova pochi micron sopra il fondo del piatto, che facilita il rinnovo della soluzione di perfusione. </li><li> Per catturare un grande campo di vista (800 x 800 micron, circa un terzo della superficie fetta del nodo), raccogliere le immagini utilizzando una lente a 10x obiettivo a secco. Abbiamo scoperto che il 10x Nikon S fluoro 0,5 NA era più adatto per le nostre analisi. </li></ol> Un tipico time-lapse esperimento di imaging ottico cattura 5 piani che coprono una profondità totale di 50 micron in (z) dimensione assiale. Fluorescenti le cellule T sono di solito ripreso ad intervalli che vanno 10 a 30 secondi per 10 a 20 min. Imaging cellule T con un microscopio confocale in posizione verticale Il microscopio confocale utilizzato in questo protocollo è un SP5 Leica dotato di un obiettivo 20x immersione in acqua (Olympus, 20x/0.95 NA). <ol start="6"><li> Fissare la sezione per la fase di imaging del microscopio. Nota: solitamente una rondella sulla preparazione per immobilizzarla. </li><li> Impostare una sessione di imaging, raccogliendo una serie di immagini lungo l&#39;asse z.. Alle cellule T immagine in una struttura di tessuto intatto, la posizione di partenza è di solito impostato a 5 a 10 micron di sotto della prima cellula T etichettati. </li></ol> 6. Rappresentante risultati Seguendo questo video-protocollo si dovrebbe aspettare di visualizzare un gran numero di cellule T fluorescenti accumulate nella zona T del nodo, un fenomeno che si verifica normalmente in questo particolare ambiente in vivo (Figura 1). In particolare, le cellule T dovrebbe essere esclusa dalle zone delle cellule B, che di solito sono appena sotto la capsula. Un esperimento riuscito anche portare a cellule T reclutati nel tessuto (Figura 2), mostrando un comportamento altamente mobili (Figura 3) in linea con i risultati pubblicati ottenuti nei linfonodi intatti 4. In media, la velocità media delle singole cellule T in fette devono essere vicini ai 10 micron / min (Figura 4). <img src="/files/ftp_upload/3054/3054fig1.jpg" alt="Figura 1"/> Figura 1. Le cellule T si accumulano in una fetta linfonodo. Fluorescenza marcata con cellule T (CMFDA, verde) sono stati aggiunti a un linfonodo fetta 30 minuti prima acquisizione delle immagini (foto in alto). L&#39;immagine è la proiezione massimo di 5 immagini coprono 50 micron in direzione z. L&#39;immagine in campo chiaro è mostrato in basso. Le immagini sono state catturate con un microscopio widefield. <img src="/files/ftp_upload/3054/3054fig2.jpg" alt="Figura 2"/> Figura 2. Le cellule T sono reclutati in una fetta linfonodo. Fluorescenza marcata con cellule T (CMFDA, verde) sono stati aggiunti a un linfonodo fetta 30 minuti prima di acquisizione delle immagini utilizzando un microscopio confocale. Le immagini sono state catturate in superficie tagliata (foto in alto) e 40 micron al di sotto (foto in basso). <img src="/files/ftp_upload/3054/3054fig3.jpg" alt="Figura 3"/> Figura 3. Le cellule T sono estremamente mobili all&#39;interno di una fetta linfonodo. Fluorescenza marcata con le cellule T sono state ripreso durante 12 minuti utilizzando un microscopio widefield. Traiettorie di singole cellule T vengono visualizzati come colori tracce di rappresentare spostamenti crescente dal blu (bassa cellule mobili) a (cellule di alta mobili) rosso. Tracce sono state calcolate utilizzando il software Imaris. La linea bianca segue il bordo del nodo, mentre l&#39;ovale tratteggiato delimita la zona delle cellule B putativo. <img src="/files/ftp_upload/3054/3054fig4.jpg" alt="Figura 4"/> Figura 4. All&#39;interno di una fetta dei linfonodi, la velocità delle cellule T è vicino ai 10 micron / min. Le velocità sono state calcolate utilizzando il software Imaris dalle tracce rappresentato in figura 3.

<ol>
	<li>Asperti-Boursin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCR7+ligands+control+basal+T+cell+motility+within+lymph+node+slices+in+a+phosphoinositide+3-kinase-independent+manner.">CCR7 ligands control basal T cell motility within lymph node slices in a phosphoinositide 3-kinase-independent manner.</a> J Exp Med. 204, 1167-1167 (2007).</li><li>Bhakta, N. R., Oh, D. Y., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+oscillations+regulate+thymocyte+motility+during+positive+selection+in+the+three-dimensional+thymic+environment.">Calcium oscillations regulate thymocyte motility during positive selection in the three-dimensional thymic environment.</a> Nat Immunol. 6, 143-143 (2005).</li><li>Matheu, M. P., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+CD4++T+cells+from+mouse+lymph+nodes+using+Miltenyi+MACS+purification.">Isolation of CD4+ T cells from mouse lymph nodes using Miltenyi MACS purification.</a> J Vis Exp. , (2007).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1869 (2002).</li><li>Ehrlich, L. I., Oh, D. Y., Weissman, I. L., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+contribution+of+chemotaxis+and+substrate+restriction+to+segregation+of+immature+and+mature+thymocytes.">Differential contribution of chemotaxis and substrate restriction to segregation of immature and mature thymocytes.</a> Immunity. 31, 986-986 (2009).</li><li>Gollmer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL21+mediates+CD4++T-cell+costimulation+via+a+DOCK2/Rac-dependent+pathway.">CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway.</a> Blood. 114, 580-580 (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

Abbiamo descritto una semplice, tecnica veloce e robusto per la produzione di fette di linfonodi, che sono utilizzati per studiare il comportamento delle cellule T introdotto. Negli ultimi anni, questo metodo è stato applicato con successo con timo e linfonodi fette di identificare i fattori extracellulari il controllo del posizionamento e della motilità delle cellule T 1,5. E &#39;stato anche utilizzato per misurare Ca 2 + risposte in timociti durante la selezione positiva e nelle cellule T sul riconoscimento dell&#39;antigene 2,6. Il test fetta sovrapposizione presenta vantaggi e limitazioni che meritano di essere discussi. Di nota, questo sistema consente l&#39;accesso al tessuto, utile se si ha la necessità di manipolare fette farmacologicamente in modo da interferire con il controllo molecolare della migrazione cellulare. A seguito di imaging, le fette possono essere trattati per immunoistochimica per raccogliere ulteriori informazioni sulle strutture che sono state fotografato. Inoltre, l&#39;osservazione può essere fatta con un tradizionale microscopio a fluorescenza widefield. Anche se la risoluzione non è buono come con un confocale o un microscopio a due fotoni e che fototossicità in linea di principio più grave con un solo fotone che con la microscopia a due fotoni, ha i vantaggi di semplicità, un costo inferiore, e più grande scelta di lunghezze d&#39;onda di eccitazione. L&#39;imaging di cellule T in un linfonodo intatto richiede l&#39;iniezione endovenosa di linfociti etichettati che successivamente a casa per gli organi linfoidi. Come abbiamo mostrato in precedenza 1, il test fetta è perfettamente compatibile con l&#39;esperimento di trasferimento adottivo. Tuttavia, il tempo necessario per le cellule T a casa nei linfonodi può complicare l&#39;uso di coloranti fluorescenti che hanno la tendenza a fuoriuscire delle cellule nel corso del tempo. Questo è il caso del Ca 2 + colorante Fura-2. Con il reclutamento veloce (&lt;30 min) di cellule T nel tessuto, il saggio fetta offre l&#39;opportunità di utilizzare tali coloranti. Infine, questo metodo ha il grande vantaggio di analizzare le funzioni delle cellule T in diversi tessuti umani tenuti in vita. Questo sistema sperimentale presenta anche dei limiti che devono essere tenuti in mente. Danni associati con le operazioni di affettamento può influire sul funzionamento delle cellule T, specialmente nella regione superficiale del tessuto vicino alla superficie di taglio. Al fine di valutare la morte delle cellule all&#39;interno della fetta, abbiamo usato il colorante fluorescente verde SYTOX che penetra le cellule con membrana plasmatica compromessa. I nostri esperimenti rivelano che circa il 20% del totale delle cellule nodali, per lo più localizzate nella regione superficiale del tessuto, sono state marcate con il colorante nucleare. Un altro problema potenziale con il test è la capacità delle fette di conservare importanti fattori solubili tra cui chemochine. Anche se abbiamo alcuna indicazione che i nostri dati sono stati colpiti da problemi di questo tipo dato che le cellule T visualizzare una buona mobilità all&#39;interno della fetta, vorremmo sottolineare l&#39;importanza di imaging cellule T nelle regioni sano situato a qualche decina di micron dalla superficie tagliata. Considerando che questo potrebbe essere fatto con microscopi tradizionali (widefield o confocale), come in questo protocollo, è probabile che il nodo preparazione linfa fetta combinato con due fotoni di imaging aumenta la risoluzione spaziale in profondità e ridurre la fototossicità.

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> Microscopio confocale </td><td> Leica </td><td> SP5 </td><td></td></tr><tr><td> Vibratome </td><td> Leica </td><td> VT1200S </td><td></td></tr><tr><td> Pinza multa </td><td> Mondo Strumenti di precisione </td><td> 14142 </td><td></td></tr><tr><td> 30 inserti Cultura millimetri </td><td> Millipore </td><td> PICM0RG50 </td><td></td></tr><tr><td> RPMI </td><td> Invitrogen </td><td> 61870010 </td><td> Completo RPMI-media è fatta aggiungendo il 10% di siero di siero fetale bovino e penicillina / streptomicina </td></tr><tr><td> Sale Hanks &#39;soluzione equilibrata (HBSS) </td><td> Invitrogen </td><td> 14170088 </td><td></td></tr><tr><td> Bassa temperatura di gelificazione agarosio, tipo VII-A </td><td> Sigma-Aldrich </td><td> A0701 </td><td></td></tr><tr><td> Butile Colla cianoacrilato, Vetbond </td><td> 3M </td><td> 1469 </td><td></td></tr><tr><td> Rondelle in acciaio inossidabile, 4 mm di diametro interno </td><td> Bricolage </td><td></td><td></td></tr><tr><td> Cellula Tracker verde CMFDA </td><td> Invitrogen </td><td> C7025 </td><td></td></tr></tbody></table>

ex vivo imaging of t cells in murine lymph node slices with widefield and confocal microscopes

Naïve cellule T in continuo traffico di organi linfatici secondari, compresi i linfonodi periferici, per individuare rari antigeni espressi. La migrazione delle cellule T nei linfonodi è un processo complesso che coinvolge sia fattori chimici tra cui cellulari e chemochine. Recentemente, l&#39;uso di microscopia a due fotoni ha permesso di tracciare le cellule T nei linfonodi intatti e per ricavare alcune informazioni quantitative sul loro comportamento e le loro interazioni con altre cellule. Mentre ci sono evidenti vantaggi di un sistema in vivo, questo approccio richiede una strumentazione complessa e costosa e fornisce un accesso limitato al tessuto. Per analizzare il comportamento delle cellule T all&#39;interno di linfonodi murini, abbiamo sviluppato un saggio fetta 1, originariamente istituito da neurobiologi e recepita di recente per murini timo 2. In questa tecnica, fluorescente cellule T sono placcati in cima ad una fetta linfa acutamente preparato nodo. In questo video-articolo, la localizzazione e la migrazione delle cellule T nel tessuto vengono analizzati in tempo reale con un widefield e un microscopio confocale. La tecnica che integra in vivo microscopia a due fotoni offre un approccio efficace per l&#39;immagine delle cellule T nel loro ambiente naturale e per chiarire i meccanismi alla base della migrazione delle cellule T.

Watch this Scientific Journal Video about Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes at JoVE.com

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Questo protocollo descrive un metodo per le cellule T immagine fluorescente introdotto a fette linfonodo. La tecnica permette in tempo reale analisi della migrazione delle cellule T con widefield tradizionali microscopi a fluorescenza o confocale.

Imaging ex vivo di cellule T nel Fette murini Linfonodo con grandangolari e microscopi confocale

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

ex-vivo-imaging-t-cells-murine-lymph-node-slices-with-widefield

Research

JoVE Journal

Immunology and Infection

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

16.7K Views.  Université Paris Descartes, CNRS (UMR 8104). Questo protocollo descrive un metodo per le cellule T immagine fluorescente introdotto a fette linfonodo. La tecnica permette in tempo reale analisi della migrazione delle cellule T con widefield tradizionali microscopi a fluorescenza o confocale.

Video: Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Trasduzione retrovirale di recettori delle cellule T nel topo le cellule T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Ricerca

Immunologia e infezioni

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Microscopia intravitale del linfonodo inguinale

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Murine Superficial Lymph Node Surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

Murine superficiale Lymph Chirurgia Node

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Imaging intravitale del Nodo poplitea Lymph mouse

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

Generazione di Linfonodo grassi Pad Chimere per lo studio del linfonodo stromale origine cellulare

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Isolamento di cellule murine Linfonodo stromali

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Un<em&gt; Ex vivo</em&gt; Modello di un attacco T-Cell Oligodendrocyte-diretto nel acute cervello fette

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

Ex Vivo infezione di Murine epidermide con Herpes Simplex Virus di tipo 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

Isolamento e<em&gt; Ex Vivo</em&gt; Cultura di Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; Celle γδ T, un Extrathymic αβT-cellule progenitrici

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

Murino linfociti Etichettatura da<sup&gt; 64</sup&gt; Cu-anticorpo recettore Targeting per<em&gt; In Vivo</em&gt; Cell Traffico da PET / CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...