The authors thank Sanjiv Luther and colleagues for helpful discussions in establishing the current lymph node digestion protocol. This work was supported by SNF grants PPOOA-_119204 and PPOOP3_144918 to S.W.R.

In questa pubblicazione video e protocollo, sono state effettuate tutte le procedure sugli animali conformemente al protocollo animali approvato dalla Autorità cantonale di Basilea Città, Svizzera. 1 Linfonodi Preparazione e digestione <ol><li> Acqua preriscaldo in un becher da 37 ° C su un agitatore magnetico con piastra di riscaldamento. </li><li> Preparare Base Medio come segue: terreno DMEM (senza piruvato) supplementato con FCS 2%, 1,2 mM CaCl 2 e Pen / Strep (100 unità di penicillina, 100 mg di streptomicina). </li><li> Sterilizzare tutti gli strumenti di dissezione prima dell&#39;uso. </li><li> Euthanize topi linfonodale donatori per CO 2 asfissia e asettico sezionare i linfonodi. Non sezionare il grasso circostante. NOTA: Questo protocollo è ottimizzato per il nodo periferico-pelle drenante linfatico (inguinale, brachiale, ascellare). </li><li> Mettere i linfonodi in una capsula di Petri sterile contenente 2 ml di ghiaccio freddo ambiente basico. </li><li> Interrompere il linfonodo capsule utilizzando due aghi 25 G fissi sulla siringa da 1 ml. </li><li> Trasferire il tessuto linfonodale interrotto in 5 ml in polipropilene tubo a fondo rotondo contenente 750 ml terreno base integrato con 1 mg / ml di collagenasi IV e 40 mcg / ml I. DNAse </li><li> Aggiungere un agitatore magnetico sterile in ciascuna provetta. </li><li> Inserire il tubo nel bicchiere con 37 ° C preriscaldato acqua e mescolare i tubi a bassa velocità (1 round / sec) per 30 min. </li><li> Rimuovere il tubo dalla agitatore magnetico con piastra di riscaldamento e lasciare frammenti linfonodali stabilirsi. </li><li> Rimuovere con cautela il surnatante arricchito in &quot;cella non stromali&quot;. NOTA: Se l&#39;analisi di T, B, cellule dendritiche e CD45 - gp38 - CD31 - è previsto, salva la frazione &quot;cellule non-stromali&quot;. </li><li> Lavare restante tessuto linfonodale una volta con 750 microlitri di media di base. NOTA: Questo passaggio è necessario solo se si lavora con i topi immuno-competenti. </li><li> Lasciate frammenti linfonodali stabilirsi. </li><li> Rimuovere ilfrazione di cellule-floating non-stromale. </li><li> Aggiungi ai frammenti linfonodali 750 microlitri terreno base integrata con 3,5 mg / ml Collagenase D e 40 mcg / ml DNasi I. </li><li> Collocare il tubo indietro nel bicchiere contenente la 37 ° C preriscaldato acqua. </li><li> Digest nodo tessuto linfatico per 5 minuti mentre lentamente mescolando. </li><li> Disaggregare linfatici frammenti di tessuto nodo di pipettaggio e mescolando 700 ml per 10 cicli a velocità massima utilizzando una pipetta multicanale automatizzata. NOTA: Questo sconvolge nodo tessuto linfatico per migliorare la digestione. </li><li> Posizionare il tubo di nuovo nel bicchiere con 37 ° C acqua preriscaldata. </li><li> Digest frammenti di tessuto linfonodale per altri 10 min, mescolando lentamente. </li><li> Disaggregare linfatici frammenti di tessuto nodo pipettando e miscelazione per 99 cicli a velocità massima utilizzando una pipetta multicanale automatizzata. </li><li> Aggiungere 7,5 ml di 0,5 M EDTA per assicurare il mantenimento della sospensione singola cellula. </li><li> Frammenti di tessuto linfonodale disaggregare per tubotting e miscelazione per 99 cicli a velocità massima utilizzando una pipetta multicanale automatizzata. </li><li> Aggiungere 750 microlitri di media di base e passare le cellule attraverso una maglia di nylon di 70 micron. </li><li> Sospensione cellulare Centrifugare 5 min a 1.500 xg, a 4 ° C. </li><li> Cellule stromali possono ora essere utilizzati per ulteriori analisi. </li></ol> 2. colorazione di cellule stromali Linfonodo <ol><li> Utilizzare una combinazione di anti-CD45, anti-podoplanin (gp38), anticorpi anti-CD31 per riconoscere con successo le cellule TRC, LEC, BEC e DN. </li><li> Incubare il tessuto linfonodale digerito con Live / inseguitore Morto, anti-CD45-FITC (1: 200), anti-gp38-PE (1: 200), anti-CD31-APC (1: 200) in 100 microlitri HBSS contenente 2 % FCS per almeno 20 min, 4 ° C, al buio. </li><li> Lavare le cellule aggiungendo 500 l di HBSS contenente 2% FCS </li><li> Sospensione cellulare Centrifugare 3 min a 1500 xg, a 4 ° C. </li><li> Risospendere in 100 ml di HBSS contenente 2% FCS. </li><li> Eseguire le cellule colorate in un citofluorimetro equipped con le seguenti ottiche: fonte di eccitazione fino a tre laser: blu (488 nm, raffreddato ad aria, 20 mW a stato solido), rosso (633 nm, 17 mW HeNe), e viola (405 nm, 30 mW a stato solido) . </li><li> Porta CD45 - cellule di escludere le cellule ematopoietiche. </li><li> Canottiere Cancello (FSC-W) e le cellule vive (live / inseguitore DEAD) per escludere doppietti e cellule morte. </li><li> Trama gp38 vs CD31 per visualizzare TRC (gp38 + CD31 -), LEC (gp38 + CD31 +), BEC (gp38 - CD31 +) cellule (vedi Figura 1) e DN (gp38 - - CD31). </li></ol>

<ol>
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The authors declare they have no competing financial interests.

Lo studio delle cellule stromali linfonodali recentemente è diventato un focus di ricerca a causa dello sviluppo di due protocolli di digestione pubblicati 6,13. Entrambi i protocolli sono adeguate per ottenere cellule stromali nodo singolo linfatici ma differiscono per l&#39;uso di enzimi digestivi e il tempo di digestione. Poiché le cellule stromali e loro marcatori di superficie sono sensibili alla digestione enzimatica e sollecitazioni meccaniche, è necessario un protocollo ottimizzato. L&#39;isolamento delle cellule stromali linfonodo vitali dalle linfonodo appena sezionato è il primo passo per eseguire analisi fenotipica e funzionale. Pertanto, è importante digerire accuratamente con la concentrazione definita di enzimi, ad una temperatura stabile e per il tempo indicato. Sono stati descritti due protocolli di digestione 6,13, ma con mineralizzazione piuttosto lunghi. Per ridurre il tempo di digestione, disaggregazione meccanica era inclusa e standardizzato con una pipetta multicanale. Su<html> e dei vantaggi del protocollo attuale è che permette l&#39;isolamento di cellule stromali in soli 45 minuti, quindi riducendo al minimo il tempo di incubazione con gli enzimi digestivi. Inoltre, agitazione costante durante la digestione permette l&#39;accesso ottimale degli enzimi digestivi alla struttura linfonodo. Inoltre, il volume di digestione è stata ridotta a 750 microlitri al minimo la quantità di enzima impiegato. Forza meccanica eccessiva distrugge giunzioni strette ma potrebbe causare la morte cellulare allo stesso tempo. Per questo motivo abbiamo testato diverse combinazioni di cicli di ri-sospensione e diversi dispositivi di dissociazione. Abbiamo trovato che l&#39;uso di una pipetta multicanale automatizzata consente la dissociazione entro 2 applicazioni di 99 cicli; applicare una pressione costante alle cellule e questo si tradurrà in sospensione di cellule singole con redditività ottimale. Quattro a sei campioni possono essere mescolati e pipettati allo stesso tempo riducendo il tempo di linfonodo isolamento delle cellule stromali. ontent &quot;&gt; Il protocollo digestione deve mantenere l&#39;espressione di superficie di quasi tutte le molecole;. solo così la colorazione concomitante con diversi marcatori può rivelare se una popolazione è omogenea o eterogenea Ad esempio, l&#39;uso di BP-3 e CD35 nella TRC gp38 + CD31 -. consente la visualizzazione e l&#39;isolamento di midollare TRC (Sanjiv Lutero, comunicazione personale) Inoltre, la frazione poco caratterizzato DN è stato ulteriormente caratterizzato come contenente + cellule e periciti Aire come cellule positive per l&#39;integrina α7 (recensione a 7, 13). Pertanto, il presente protocollo è stato confrontato con quelli pubblicati. Come mostrato nei risultati, le frequenze e il numero di cellule stromali linfonodali simili sono stati ottenuti con il protocollo in corso rispetto a quelli pubblicati. interessante notare che, la frequenza e il numero di isolati TRC sono stati più alta utilizzando il protocollo Fletcher suggerendo una migliore dissociazione delle strutture TRC rispetto al protocollo attuale eProtocollo Link. Inoltre, l&#39;espressione di diverse molecole di superficie è stata arricchita con il protocollo corrente rispetto al protocollo Fletcher. Queste differenze nel metodo di digestione potrebbe essere il motivo per leggermente diversi pattern di espressione negli studi sulle cellule stromali linfonodali 6,19,21. Potrebbe essere utile per testare diversi protocolli di digestione a causa dei vantaggi di ogni protocollo. Il protocollo attuale potrebbe essere migliore per l&#39;analisi di espressione di superficie, mentre il protocollo Fletcher potrebbe essere utile per l&#39;isolamento di un numero elevato di cellule vitali per l&#39;analisi di trascrizione (come pubblicato in Malhotra et al. 7). Pertanto, confrontando tutti e tre i protocolli di digestione potrebbe essere importante per ottenere risultati ottimali dopo l&#39;isolamento delle cellule stromali linfonodali e dovrebbe essere visto come complementare.

I linfonodi sono compartimenti specializzati in cui vengono avviate e coordinate risposte immunitarie adattative contro stranieri e di auto-antigeni. La procedura qui presentata descrive una breve digestione enzimatica combinata con pipettaggio meccanico automatizzato per ottenere linfonodo sospensione singola cella e ottenere l&#39;accesso a linfonodali cellule stromali vitali che mantengono l&#39;espressione di superficie di diverse molecole. Formare cellule stromali linfonodale l&#39;impalcatura del linfonodo e compiere tre funzioni principali: primo filtrano fluidi corporei di assaggiare antigeni, gli agenti patogeni e il loro modello molecolare associata patogeni (PAMP), così come le citochine e pericolo del modello molecolare associato (smorza) presente nel corpo. In secondo luogo, si attraggono e istruiscono le cellule presentanti l&#39;antigene (APC) e linfociti di interagire e di avviare le risposte immunitarie adattative; e terzo, offrono un ambiente strutturale per l&#39;omeostasi e la differenziazione dei linfociti 1-3. Durante le cellule stromali infiammazione dei linfonodi producono fattori di crescita, citochine e chemochine, adattarsi al rigonfiamento in tal modo organizzare l&#39;interazione tra cellule dendritiche (DC), T, e B- cellule. L&#39;orchestrazione delle risposte immunitarie è possibile solo a causa della complessa architettura strutturale formato da diverse popolazioni di cellule stromali. Cellule stromali linfonodi sono cellule CD45 negativi e si distinguono per l&#39;espressione di CD31 o gp38 in cellule endoteliali e fibroblastiche 1 -6. Gp38 + CD31 - definisce le cellule della zona T reticolari (TRC, noto anche come FRC: cellule reticolari fibroblastiche), gp38 + CD31 + definisce cellule linfatiche endoteliali (LEC), gp38 - CD31 + definisce cellule endoteliali del sangue (BEC). Inoltre, la caratterizzazione delle sottopopolazioni ha rivelato l&#39;esistenza di altre cellule stromali linfonodali. Infatti, una piccola popolazione di cellule periciti-simile è stato caratterizzato nellagp38 - CD31 - la popolazione 7. Pertanto, l&#39;adattamento della procedura di isolamento è vantaggiosa per l&#39;identificazione e la caratterizzazione delle proprietà funzionali di varie cellule stromali linfonodali. Prima dello sviluppo del linfonodo cellule stromali digestione protocolli di studio delle cellule stromali linfonodali era limitato a osservazioni in situ utilizzando la sezione di tessuto e microscopia. Tuttavia, studi strutturali e funzionali hanno mostrato caratteristiche importanti delle cellule stromali linfonodali. Cellule stromali linfonodi sono associati con podoplanin, collagene e della matrice extracellulare (ECM) proteine ​​per formare un complesso di 3 struttura dimensionale chiamato sistema conduit, che trasporta le proteine ​​linfatiche e massa molecolare associato-basso dal seno subcapsulare di linfonodo all&#39;alta endoteliale venule nella T cellule zona 8. Dendritiche sono in stretto contatto con le cellule dello stroma e può osservare sporge nel con tubolarestruttura duit di campione di fluido e rilevare gli antigeni 8. L&#39;interazione delle cellule stromali linfonodali (TRC e LECs) con DC è mediata dal rilascio e la presentazione delle chemochine CCL21 e CCL19 9,10. CCL19 e CCL21 sono riconosciuti dal recettore CCR7 facilitare dendritiche e le cellule T di migrare al linfonodo zona di cellule T 4,11. Nonostante l&#39;uso di chemochine simili, le cellule DCs e T hanno diverse rotte migratorie verso i linfonodi 12. Successivamente, utilizzando la digestione enzimatica del linfonodo e l&#39;isolamento delle cellule stromali linfonodali puri, studi funzionali sono stati condotti sul ruolo delle diverse cellule dei linfonodi stromali e la loro capacità di interagire con le cellule dendritiche e le cellule T / B 6,13. In primo luogo, il crosstalk tra la produzione di cellule T effettrici IFN-γ e cellule stromali linfonodali induce la produzione del metabolita ossido nitrico dimostrato di smorzare le risposte delle cellule T e la proliferazione negli organi linfoidi secondari 14-16. In secondo luogo, linfa ncellule stromali ODE sono stati riportati per supportare la differenziazione delle sottopopolazioni DC regolamentazione attraverso la produzione di IL-10 17, e di modulare naif omeostasi delle cellule T attraverso la produzione di IL-7 6,18. In terzo luogo, espressione di TLR in cellule stromali linfonodali suggerisce che le cellule stromali sono suscettibili di segnale derivato da una infezione o di auto-molecole rilasciato durante lesioni dei tessuti. Infatti, il trattamento di cellule stromali linfonodali con il ligando di TLR3 poly (I: C) induce una modesta sovraregolazione di grande espressione di istocompatibilità di classe I e sovraregolazione di co-inibitorio molecola PD-L1, ma non di molecole costimolatorie, con conseguente drammatici cambiamenti nel tessuto periferico antigeni espressione 19. Diversi gruppi hanno mostrato cellule stromali linfonodali esprimono antigeni tissutali periferiche e inducono tolleranza delle cellule T autoreattive 19,21-27. Pertanto, la comprensione delle interazioni tra cellule stromali linfonodali e l&#39;altro re migratorio ecellule linfonodali sidente aiuteranno a trovare nuove molecole bersaglio per consentire l&#39;attivazione o la soppressione delle risposte immunitarie durante l&#39;infiammazione. Pertanto, è necessaria l&#39;attuazione della separazione enzimatica pubblicata del linfonodo. Protocolli precedentemente pubblicati utilizzano diverse combinazioni di base di collagenasi-digestione enzimatica con una bassa sollecitazione meccanica 6,19,20. Tuttavia, lunghe incubazioni con enzimi di digestione o la diversa combinazione di digestione enzimatica potrebbero degradare diverse molecole di superficie necessaria per analizzare lo stato di attivazione e di individuare nuove cellule stromali linfonodali. A seconda del tipo di analisi delle cellule stromali, il protocollo Link o Fletcher protocollo potrebbe essere più adatto. Nella procedura descritta, una digestione enzimatica leggermente più corto è combinato con disaggregazione meccanica automatizzata per ridurre al minimo la superficie marcatore degradazione delle cellule stromali nodo linfa vitale. Questa procedura consente di isolamento altamente riproducibile edistinzione dei linfonodi popolazioni di cellule stromali con bassa variabilità e di oltre il 95% vitalità. Le cellule stromali linfonodi isolati a fresco possono essere utilizzati direttamente per l&#39;espressione marcatore di superficie, analisi delle proteine, e gli studi trascrizionali, nonché creazione di linee di cellule stromali di effettuare saggi funzionali in vitro.

<table border="0" cellpadding="0" cellspacing="0" width="959">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:231px;">LifeTechnologies-Gibco</td>
			<td style="width:119px;">41965-039</td>
			<td style="width:395px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FCS</td>
			<td style="width:231px;"></td>
			<td style="width:119px;"></td>
			<td>Final concentration 2%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:119px;">499609</td>
			<td>Final concentration 1.2 mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Collagenase IV</td>
			<td style="width:231px;">Worthington Biochemical Corporation</td>
			<td style="width:119px;"></td>
			<td>&#62;160 units per mg dry weight, use at final concentration of 1 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase D</td>
			<td style="width:231px;">Roche</td>
			<td style="width:119px;">11088882001</td>
			<td>use at 3.5 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNAse I</td>
			<td style="width:231px;">Roche&#160;</td>
			<td style="width:119px;">11284932001</td>
			<td>use at 40 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">stiring magnets</td>
			<td style="width:231px;">FAUST</td>
			<td style="width:119px;"></td>
			<td>5 mm long-2 mm &#248;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polystyren Round-Bottom Tubes 5ml</td>
			<td style="width:231px;">Falcon-BD Bioscience</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Magnetic stirrer with heating funktion</td>
			<td style="width:231px;">IKA-RCT-standard</td>
			<td style="width:119px;">9720250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petridishes 100 mm, sterile</td>
			<td style="width:231px;">TPP</td>
			<td style="width:119px;">6223201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25G needles</td>
			<td style="width:231px;">Terumo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD45 Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 30-F11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD11c Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone N418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse Podoplanin Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 8.1.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD31 Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone MEC13.3</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;"> 
			Eppendorf Xplorer plus, Multichannel</td>
			<td style="width:231px;">Eppendorf</td>
			<td style="width:119px;">4861 000.821/830</td>
			<td>1.250 &#181;l max. volume</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD140a</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone APA5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD80</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 16-10A1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD40</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 1C10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse I-Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone AF6-120.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD274 (PD-L1)</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 10F.9G2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LIVE/DEAD Fixable Near-IR Dead Cell stain kit</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:119px;">L10119</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Il presente protocollo è un protocollo di digestione modificata pubblicato da Link et al., 2007 6 con un tempo di digestione breve (45 minuti massimo) a causa di disaggregazione meccanica con una pipetta multicanale automatizzata. Inoltre, la procedura è più standardizzato, minimizza la degradazione dei marcatori di superficie su diverse cellule stromali linfonodali e permette la gestione di più di un campione allo stesso tempo. Collagenase IV e Collagenase D in Links protocollo 6 e protocollo corrente o Collagenase P e dispasi in protocollo Fletchers 13 mantengono linfonodo cellule stromali vitalità e risparmiano la digestione di alcuni marcatori di superficie, tra cui CD45, CD31 e gp38. Per testare i nodi di protocollo, ascellari, brachiale e linfonodi inguinali attuali sono stati rimossi e digeriti per isolare le cellule stromali. Analisi di CD45, gp38 e CD31 espressione dimostrato la presenza di cellule TRC, LEC, BEC e DN isolati da C57BL / 6 mghiaccio (Figura 1, strategia di gating). Lo stress meccanico potrebbe ridurre la vitalità delle cellule stromali nodo linfatiche durante la digestione. Confrontando la fattibilità di sottopopolazioni isolate di cellule stromali utilizzando i tre diversi protocolli, si è constatato che la redditività è stata superiore al 95% nel protocollo Fletcher, ma inferiore sia per il protocollo corrente e il protocollo di collegamento (Figura 2A), anche se corrente spettacolo protocollo migliore sopravvivenza nella sottopopolazione BEC poi Link Protocol. Il numero di cellule totali recuperati dopo la digestione dei linfonodi sottopopolazioni di cellule stromali è stata leggermente superiore nel protocollo attuale rispetto al protocollo di collegamento e protocollo Fletcher (Figura 2A). Questi risultati dimostrano che il protocollo attuale mantiene la vitalità delle cellule stromali linfonodali simili a protocolli pubblicati. Le principali differenze tra i tre protocolli sono elencati nella Tabella 1. Marcatori di superficie di un<html> re necessarie per caratterizzare le cellule stromali linfatiche nodo mediante citometria di flusso e di isolarli dai ordinamento delle cellule. TRC e LEC isolato via corrente, sono stati confrontati i protocolli di collegamento e Fletcher per IA b, CD140a, CD80, PD-L1 ed espressione CD40. Dopo aver isolato cellule stromali da tutti e tre i protocolli, abbiamo trovato l&#39;espressione di IA B, CD80, CD140a, PD-L1, e CD40 era più alta su digestione con CP e LP in entrambi TRC e LEC (Figura 2b). Questi risultati suggeriscono che il degrado di alcune molecole di superficie con collagenasi IV e D sono meno forti che con Collagenase P e dispasi. Nel loro insieme, l&#39;attuale protocollo include una breve digestione combinata con disaggregazione meccanico automatizzato per ridurre al minimo la superficie marcatore degradazione delle cellule stromali nodo linfa vitale. &quot;/&gt; Figura 1 nodi TRC, cellule LEC, BEC, e DN colorazione, gating, e quantificazione. Linfa C57BL / 6 topi sono stati digeriti seguendo il protocollo attuale e colorati con CD45, CD31 e gp38, Live / Morto per definire TRC, LEC, BEC, e DN cellule mediante citometria a flusso. I dati mostrano risultati rappresentativi della colorazione. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51803/51803fig1highres.jpg" target="_blank">Clicca qui per vedere l&#39;immagine ingrandita.</a> <img alt="Figura 2" fo:content-width="5in" src="/files/ftp_upload/51803/51803fig2highres.jpg" width="500" /> Figura 2 Viabilità e superficiale espressione marker di cellule stromali linfonodali. Linfonodi di topi C57BL / 6 sono stati digeriti in seguito alla corrente (CP), il protocollo di Fletcher (FP) e colorati con CD45, gp38 e CD31 di Link (LP) e per definire TRC, LEC, BEC e DN cellule mediante citometria a flusso. A) Viabillità e il numero totale delle cellule di tutte le sottopopolazioni di cellule stromali linfonodo dopo Live / colorazione Morto (n ≥ 5, i dati combinati di due esperimenti indipendenti, barre rappresentano SEM). B) Analisi di espressione di superficie di IA b (APC-Cy7), CD140a (PerCP-Cy5.5), CD80 (PerCP-Cy5.5), PD-L1 (PE-Cy7) e CD40 (PE-Cy7) su TRC e LEC (n = 6 per FP e n = 11 per LP e CP , dati aggregati di 2 esperimenti indipendenti, barre rappresentano SEM). Geometriche delle IFM valori di auto-fluorescenza per LEC-APC-Cy7: 324 ± 97, LEC-PECy7: 163 ± 82,5, LEC-PerCpCy5.5: 156 ± 36, TRC-APC-Cy7: 349 ± 152, TRC-PECy7: 117 ± 54, TRC-PerCP-Cy5.5: 121 ± 45. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51803/51803fig2highres.jpg" target="_blank">Clicca qui per vedere l&#39;immagine ingrandita.</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Corrente Protocollo-Enzimi </td><td> Current-protocollo </td><td> Link Protocol-enzimi </td><td> Link-protocollo </td><td> FLetcher Protocol-enzimi </td><td> Fletcher-protocollo </td></tr><tr><td> Collagenase IV, DNasi I </td><td> 30 min, 37 ° C, agitazione </td><td> Collagenase IV, DNAsi I </td><td> 30 min, 37 ° C </td><td> Collagenase P, dispasi, DNAse I </td><td> 20 min, 37 ° C, invertire ogni 5 min </td></tr><tr><td> Collagenase D, DNasi I </td><td> 5 min, 37 ° C, agitazione, risospendere </td><td> Collagenase D, DNAsi I </td><td> 20 min, 37 ° C </td><td> Collagenase P, dispasi, DNAse I </td><td> 10 min, 37 ° C, risospendere 30 sec </td></tr><tr><td></td><td> 10 min, 37 ° C, agitazione </td><td> Collagenase D, DNAsi I </td><td> 37 ° C, risospendere ogni 10 minuti fino a completa digestione </td><td> Collagenase P, dispasi, DNAse I </td><td> 37 ° C, risospendere ogni 5 minuti fino a completa digestione </td></tr><tr><td></td><td> risospensione meccanica automatizzata </td><td></td><td></td><td></td><td></td></tr></tbody></table> Tabella 1. brevi riassunti di CP, LP, enzimi FP usati e protocolli.

Watch this Scientific Journal Video about Isolation of Murine Lymph Node Stromal Cells at JoVE.com

Isolation of Murine Lymph Node Stromal Cells

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 ...

isolation-of-murine-lymph-node-stromal-cells

Nanyang Technological University

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JoVE Journal

Immunology and Infection

31.0K Views.  University of Basel and University Hospital Basel. 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. 

Video: Isolation of Murine Lymph Node Stromal Cells

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 ...

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Immunologia e infezioni

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

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

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

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

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 ...

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 ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

Isolamento, elaborazione ed analisi dei murini gengivali

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

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, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Isolamento, identificazione e purificazione di cellule murine timica epiteliali

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Mieloide isolamento delle cellule da topo pelle e drenante linfonodi seguito intradermica immunizzazione con vivo attenuato<em&gt; Plasmodium</em&gt; sporozoiti

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

Raccolta e trattamento dei linfonodi da grandi animali per l'analisi del RNA: preparazione per gli studi di trascrittomica di linfonodo di grandi specie animali

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

Determinazione dei sottoinsiemi di regolamentazione delle cellule T nel timo murino, pancreatico drenante linfonodi e milza mediante citometria a flusso

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...