Lo studio è sostenuto dal Ministero della Pubblica Istruzione, Singapore (ACRF Tier1-RG40 / 13 e Tier2-MOE2013-T2-2-038). Il manoscritto è stato curato da Amy Sullivan da Obrizus Communications.

Tutti i topi sono allevati e mantenuti in specifiche condizioni di assenza di patogeni e tutti i protocolli del mouse sono condotte in conformità con le linee guida del Comitato Cura e uso istituzionale degli animali. 1. Preparazione di tamponi e reagenti <ol><li> Preparare completa Roswell Park Memorial Institute (RPMI) medium (siero 10% inattivato al calore bovino fetale (FBS), 2 mM L-glutammina, penicillina (100 UI / ml) / streptomicina (100 mcg / ml), 55 mM 2-mercaptoetanolo ). </li><li> Preparare 20x soluzione salina bilanciata (BSS) 1 Stock e della 2, separatamente. <ol><li> Preparare 20x BSS magazzino 1 (111 mm destrosio, potassio fosfato 8,8 mm, 26,7 mM sodio fosfato bibasico in 1 L di acqua sterile) e aggiungere 40 ml di 0,5% rosso fenolo a 20x BSS giacenza 1 prima della messa a punto finale del volume. filtro sterile con 0,2 micron filtro prima di riporre a 4 ° C. </li><li> Preparare 20x BSS magazzino 2 (25.8 mM cloruro di calcio diidrato, cloruro di potassio 107 mM, ch 2,73 M di sodioLoride, 19.6 mM di cloruro di magnesio esaidrato, 16,6 mM solfato di magnesio in 1 L di acqua sterile). filtro sterile con 0,2 micron filtro prima di riporre a 4 ° C. </li><li> Per preparare 1x BSS per uso sperimentale, diluire 50 ml di BSS magazzino 1 e 50 ml BSS magazzino 2, separatamente, in 400 ml di acqua sterile ciascuno. Combinare entrambe le soluzioni diluite, regolare a pH 7, e aggiungere 20 ml di FBS (2%). Top fino a 1 L con acqua sterile e filtro sterile con 0,2 micron filtro. NOTA: soluzioni madri BSS devono essere preparati separatamente perché la miscelazione delle scorte BSS concentrati direttamente può provocare precipitazioni. </li></ol></li><li> Globuli rossi (RBC) Lysis Buffer <ol><li> Per preparare tampone di lisi RBC, mescolare 9 parti magazzino cloruro di ammonio (155 mm cloruro di ammonio in acqua sterile) e 1 parte di magazzino Tris-base (130 mm Tris (idrossimetil) aminomethane in acqua sterile, pH 7,65) prima dell&#39;uso. NOTA: Conservare soluzioni madre sterili di cloruro di ammonio e Tris-base a 4 ° C. Preparare lisi BUffer fresco per garantire efficiente lisi dei globuli rossi. </li></ol></li></ol> 2. Generazione di linfociti sospensione dalla milza o linfonodi NOTA: E &#39;importante preparare tutti i reagenti e le attrezzature necessarie per l&#39;esperimento prima che l&#39;eutanasia del mouse e di generare sospensioni singola cella di linfociti nel più breve tempo possibile mantenere alti Viabilità cellulari. <ol><li> Eutanasia del mouse sperimentale di dislocazione cervicale o CO 2 asfissia. NOTA: Da questo punto in poi, tutte le procedure sperimentali devono essere eseguite in modo asettico. </li><li> Immergere l&#39;intero del mouse in etanolo al 70% prima di effettuare qualsiasi incisioni. Rimuovere la milza e linfonodi asettico 8, e metterli in separati da 15 ml provette contenenti 5 ml di ghiacciata RPMI / FBS (RPMI con il 2% FBS) o BSS / FBS (dal punto 1.2.3). NOTA: Dal momento che BSS impedisce efficiente lisi RBC, usare RPMI per la preparazione della sospensione cellulare della milza e passare a BSS after RBC lisi. </li><li> Per generare una sospensione singola cella da milza o linfonodi, posizionare l&#39;organo (s) tra due pezzi di sterile 100 mesh filtro micron delle cellule in una capsula di Petri contenente 2 ml di ghiacciata RPMI / FBS o BSS / FBS. Utilizzando lo stantuffo di una siringa da 1 ml, schiacciare l&#39;organo (s) finché non è stato strappato in parti molto fini. </li><li> Trasferire la sospensione di cellule in una provetta da 15 ml e lavare il filtro cella di maglia con ghiacciata RPMI / FBS o BSS / FBS. Raccogliere la sospensione cellulare residua, aggiungerlo alla stessa tubo da 15 ml, e spin down a 453 xg per 5 minuti a 4 ° C. Rimuovere il surnatante. NOTA: risospendere le cellule in pellet muovendo il tubo con le dita prima di aggiungere buffer di lisi RBC o medio nelle fasi successive. </li><li> Preparare temperatura ambiente tampone di lisi (RT) RBC durante la centrifugazione della sospensione cellulare (vedi passo 1.3.). Dopo pellet cellule e rimuovere il surnatante, risospendere le cellule con 1 ml di tampone di lisi RBC per ogni 10 8 cellule. Incubate la reazione di lisi a temperatura ambiente per 3-4 min. </li><li> Smettere di RBC lisi con 14 ml di BSS ghiacciata / FBS e spin down a 453 xg per 5 minuti a 4 ° C. </li></ol> 3. Purificazione di B e le cellule T <ol><li> Purificazione di cellule B <ol><li> Contare le cellule utilizzando un emocitometro. Risospendere fino a 10 8 cellule della milza in 300 ml BSS / FBS e aggiungere 50 ml di anti-CD43 microsfere magnetiche 9,10. Per rimuovere le cellule morte, aggiungere 30 ml annessina V sfere magnetiche. Incubare la sospensione cellulare in frigorifero a 4 ° C per 30 min. </li></ol></li><li> Purificazione di cellule T <ol><li> Contare le cellule utilizzando un emocitometro. Risospendere fino a 10 8 cellule in non-T anticorpi deplezione delle cellule cocktail (anticorpi biotinilati contro CD19, B220, Gr-1, TCR-γδ, CD49b, CD11c, CD11b, Ter119 e CD4 o CD8 seconda popolazione di cellule bersaglio per essere purificato), diluito 1: 200 in 200 ml BSS / FBS 4,5. Incubare la sospensione cellulare in un4 ° C frigorifero per 15 min. </li><li> Dopo l&#39;incubazione, aggiungere 10 ml BSS / FBS per lavare le cellule e spin down a 453 xg per 5 minuti a 4 ° C. Rimuovere il surnatante e risospendere le cellule in 165 microlitri BSS / FBS con 30 microlitri microsfere streptavidina e 15 microlitri annessina V sfere magnetiche. Incubare la sospensione cellulare in frigorifero a 4 ° C per 30 min. NOTA: per garantire anche l&#39;etichettatura con le microsfere magnetiche, incubare le cellule con microsfere per 15 minuti, poi mescolare la sospensione cellulare con delicatezza toccando il tubo da 15 ml e incubare un altro 15 minuti durante la fase 3.1.1. o 3.2.2. </li></ol></li><li> Preparazione di separazione colonna per Cell Purificazione <ol><li> Preparare una colonna di separazione non utilizzata durante l&#39;etichettatura microperla delle cellule (fase 3.1.1. E 3.2.2.). Pre-caldo BSS (senza FBS) per RT e utilizzare 2 ml per lavare ed equilibrare la colonna in modo asettico. Dopo equilibrio con BSS, la colonna lavato non dovrebbe essere permesso di asciugare. NOTA: Usiamo i LS column invece della colonna LD consigliato a causa della sua riutilizzabilità (vedi punto 3.4.). </li><li> Dopo l&#39;etichettatura con le sfere magnetiche, aggiungere 14 ml BSS / FBS per lavare le cellule e spin down a 453 xg per 5 minuti a 4 ° C. Rimuovere il surnatante e risospendere le cellule in 1-3 ml di RT BSS. </li><li> Attaccare sterile 21 ago G alla punta della colonna di ridurre la portata durante il processo di purificazione. Caricare la sospensione cellulare sulla colonna equilibrata e raccogliere il flusso attraverso contenente cellule bersaglio purificate. NOTA: evitare l&#39;introduzione di bolle nella colonna durante il caricamento. </li><li> Lavare la colonna una volta con 1 ml BSS / FBS e raccogliere il flusso attraverso contenente le cellule bersaglio purificate. Ricarica la colonna con il flusso attraverso nuovamente. Raccogliere il flusso attraverso dopo il secondo carico nello stesso tubo 15 ml. </li><li> Lavare la colonna 3 volte con 1 ml BSS / FBS e raccogliere il flusso attraverso contenente le cellule bersaglio purificate. Successivamente, aggiungere 5 ml BSS / FBS a the colonna e, con stantuffo, lavare le cellule marcate magneticamente su colonna in un nuovo tubo 15 ml. </li><li> Controllare la purezza delle cellule raccolte mediante citometria di flusso utilizzando anticorpi che si legano alla superficie antigeni di B purificati o cellule T 4,5. </li></ol></li><li> Riutilizzare la colonna di separazione NOTA: La colonna LS può essere riutilizzato fino a 4 volte senza compromettere l&#39;efficienza di purificazione. <ol><li> Lavare la colonna 3 volte con 5 ml di tampone fosfato salino (PBS) e 3 volte con 5 ml di acqua distillata dall&#39;alto con il pistone. </li><li> Lavare la colonna con 5 ml di etanolo al 70% e asciugare la colonna ampiamente utilizzando un rubinetto dell&#39;aria per evitare la formazione di ruggine nella colonna. </li><li> Per preparare una colonna LS utilizzato per un esperimento di purificazione separata, lavare la colonna dal basso verso l&#39;alto con 5 ml di etanolo al 70% utilizzando l&#39;adattatore siringa. Successivamente, lavare la colonna dal basso verso l&#39;alto due volte con 5 ml di PBS sterile, seguiti da 5 ml di PBS volta dalla sommità della colonna. Aggiungere 2ml RT BSS per equilibrare la colonna e quindi procedere al caricamento la colonna con cellule marcate. </li></ol></li></ol> 4. CFSE Etichettatura e stimolazione NOTA: Purified cellule possono essere sottoposte ad una varietà di in vitro e in vivo saggi funzionali. Qui, usiamo le cellule T purificate per determinare la capacità di stimolazione delle cellule T di APC 5. <ol><li> soluzione Pre-caldo etichettatura (0,1% FBS in PBS) per 37 ° C prima del CFSE carico. NOTA: l&#39;utilizzo di una bassa percentuale di FBS in PBS riduce la morte delle cellule durante CFSE carico e riduce al minimo la perdita di cellule durante la centrifugazione. Tuttavia, troppo FBS possono interferire con CFSE carico. </li><li> Lavare le cellule due volte con purificate soluzione di etichettatura, poi ri-sospese a 2 x 10 7 cellule / ml in soluzione di etichettatura pre-riscaldato in una provetta da 15 ml. </li><li> Preparare 10 soluzione CFSE mM (1: 500 diluizione di 5 mm CFSE soluzione madre) in soluzione di etichettatura pre-riscaldato. soluzione CFSE dovrebbeessere preparato fresco ogni volta per ottenere l&#39;etichettatura ottimale. </li><li> Per caricare cellule con CFSE, aggiungere sospensione cellulare 1 parte a 1 parte 10 mM soluzione CFSE in una provetta da 15 ml e incubare al buio per 10 minuti a 37 ° C. Una concentrazione finale di 5 mM CFSE viene utilizzato per etichettare 1 x 10 7 cellule / ml. </li><li> Invertire il tubo ogni 2 minuti per assicurare una miscela omogenea di cellule durante CFSE carico. </li><li> Per arrestare la reazione, aggiungere diversi volumi di medie RPMI completo ghiacciata e spin down a 453 xg per 5 minuti a 4 ° C. Al successo CFSE di carico, il pellet cellulare apparirà giallastro. </li><li> Lavare CFSE caricato cellule una volta con RPMI completo ghiacciata e spin giù a 453 xg per 5 minuti a 4 ° C prima dell&#39;uso per la coltura in vitro o in vivo di stimolazione. </li></ol> 5. In Vitro Stimolazione <ol><li> Preparare una soluzione 2x magazzino di stimoli (2x stimoli soluzione stock) immediatamente prima dell&#39;uso in modo che il 100 microlitri di 2x stimoli soluzione madre può essere aggiunto a 100 ml di cellule in un volume finale di 200 microlitri per pozzetto in una piastra da 96 pozzetti. </li><li> Se è necessaria una piastra ricoperta di stimoli (IgM o CD40 per le cellule B o CD3 e CD28 per le cellule T), diluire gli stimoli in PBS e pre-coat la piastra di coltura a 4 ° C durante la notte. In alternativa, la piastra di coltura può essere rivestita a 37 ° C per 1 ora il giorno dell&#39;esperimento. Lavare la piastra rivestita due volte con PBS (Non lasciare che la piastra si asciughi in qualsiasi momento). </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Per le cellule B </td><td></td></tr><tr><td> Stimoli </td><td> concentrazione finale </td></tr><tr><td> (Ab &#39;) 2 di capra anti-topo IgM F </td><td> 0.6-2.4 mg / ml </td></tr><tr><td> Anti-topo CD40 mAb </td><td> 0,5-2 mg / ml </td></tr><tr><td> ricombinanteMouse IL-4 </td><td> 25 U / ml </td></tr><tr><td> lipopolisaccaride </td><td rowspan="2"> 0.1-10 mg / ml </td></tr><tr><td> (LPS) da E. coli sierotipo 055: B5 </td></tr><tr><td></td><td></td></tr><tr><td> Per le cellule T </td><td></td></tr><tr><td> Stimoli </td><td> concentrazione finale </td></tr><tr><td> Anti-CD3 (piatto rivestito) </td><td> 2-10 mg / ml </td></tr><tr><td> (50 ul / pozzetto per il rivestimento) </td><td></td></tr><tr><td> Anti-CD28 (piatto rivestito) </td><td> 2 ug / ml </td></tr><tr><td> Ricombinante IL-2 </td><td> 40 U / ml </td></tr><tr><td> PDBu (Phorbol estere) </td><td> 5-50 ng / ml </td></tr><tr><td> A23187 (calcio ionoforo) </td><td> 250 ng / ml </td></tr></tbody></table> Tabella 1: Concentrazioni di stimoli utilizzati per stimolare i linfociti in coltura in vitro.<ol start="3"><li> Per le cellule B CFSE-etichettati, ri-sospendere o 3 x 10 6 cellule / ml in terreno completo RPMI e cultura in triplice copia con 3 x 10 5 cellule / pozzetto in piastre da 96 pozzetti fondo piatto per 72 ore. </li><li> Per le cellule T CFSE-etichettati, ri-sospendere a 0,5-3 x 10 6 cellule / ml in terreno RPMI completo e la cultura in triplice copia con 0,5-3 x 10 5 cellule / pozzetto in piastre da 96 pozzetti a fondo sferico per 48 o 72 ore . </li></ol> 6. In Vivo Stimolazione <ol><li> Per la stimolazione in vivo, trasferimento adoptively 4 x 10 6 cellule T CFSE-etichettati per mouse (per via endovenosa (iv) in 200 ml di PBS) in ogni mouse destinatario MHC-abbinato. NOTA: In questo protocollo, le cellule T CFSE-etichettati possono essere trasferiti utilizzando adoptively vena della coda o iniezione retro-orbitale come queste cellule saranno a casa per linfoidi organi come i milza e nei linfonodi. </li><li> Sfida i topi riceventi un giorno in più con l&#39;antigene. NOTA:In questo esempio, usiamo ovalbumina (proteina OVA, 50 mg / mouse) come antigene perché OVA-specifici, recettore delle cellule T (TCR), le cellule T sono state -transgenic adoptively trasferite in topi riceventi. Preparare proteine OVA in PBS sterile e iniettare 100 ml di OVA proteine / PBS o il controllo PBS, per via sottocutanea (SI), in ogni topo ricevente 5. </li><li> Harvest e generare sospensioni singola cellula da organi linfoidi (linfonodi e milza) di topi riceventi 3 giorni dopo l&#39;immunizzazione con la proteina OVA o PBS. linfonodi separare in linfonodi prossimali (PLN), che comprende ascellare, brachiale e superficiali linfonodi cervicali) e linfonodi distali (DLN), che comprende mesenterica, poplitea, inguinali, lombari, e linfonodi caudale. cellule macchia con gli anticorpi FACS appropriate per verificare la presenza di proliferazione delle cellule T. NOTA: In questo esperimento di monitoraggio delle cellule CFSE, CFSE-etichettati cellule T da topi di controllo PBS-iniettati stabilire una linea di base per la fluorescenza non dividing cellule. Divisioni cellulari di proliferare, le cellule, CFSE-etichettati antigene-stimolata sono visualizzati misurando i picchi di fluorescenza 12,13. Con il controllo PBS, il numero di divisioni cellulari di proliferanti, cellule T CFSE etichettati può essere determinato 12,13. </li><li> Analizzare i dati di proliferazione cellulare CFSE marcato confrontando il numero di divisioni cellulari o picchi tra i campioni (figure 1 e 2). NOTA: Per esempio, CFSE marcato, le cellule T specifiche per OVA adoptively trasferite in topi riceventi ricevono l&#39;antigene OVA saranno sottoposti proliferazione attiva rispetto ai topi di controllo PBS-iniettati 5,12. Inoltre, è degno di nota sottolineare che ci sono molti modi per analizzare i dati di proliferazione cellulare CFSE, come dimostrato da Hawkins e colleghi 14. </li></ol>

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The authors have nothing to disclose.

In questo protocollo, dimostriamo una procedura per purificare i linfociti da organi linfoidi. la purificazione delle cellule usando magnetica ordinamento tallone è un metodo semplice e veloce che produce vitali, cellule bersaglio altamente purificate. I passaggi critici all&#39;interno del protocollo La vitalità cellulare e la resa delle cellule Il mantenimento di vitalità delle cellule lignaggio ematopoietiche ...

Mature lymphocytes generally exist in the resting state if there is no pre-existing infection or inflammation in the individual. Therefore, it is important to retain the na&#239;ve status of lymphocytes during the isolation process before performing in vitro or in vivo functional assays. The key to ensuring consistent and reproducible results is to limit any unnecessary manipulation of the cells. 
Magnetic cell sorting utilizes antibodies and microbeads to label cells so as to enrich the cell population of interest. With this approach, there are two purification strategies: positive enrichment and negative depletion. Positive enrichment enriches the cell population of interest using an antibody that binds to the target cells. Negative depletion, on the other hand, depletes non-target cells, leaving the cell population of interest. In our lab, we prefer negative depletion to positive enrichment because the binding of antibodies to the target cells could potentially alter cell features and behavior. In fact, many established cell surface markers suitable for the isolation of a particular cell population are also functional receptors. 
Magnetic cell sorting not only yields highly pure populations of viable target cells, it is also less time-consuming and avoids the cellular stress induced by high-pressure flow used in fluorescence-activated cell sorting (FACS). By labeling the unwanted cell populations and depleting them using a magnetic separation column, we are able to perform rapid cell isolation without compromising the viability of the target cell population. In this protocol, we demonstrate the use of negative depletion strategies to purify na&#239;ve B cells or T cells.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>RPMI 1640 (without L-Glutamine)</td><td>Gibco</td><td>31870025</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td></td><td></td><td>Heat inactivated&amp;nbsp;</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030024</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140114</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Gibco</td><td>21985023</td><td></td></tr><tr><td>Anti-CD43 magnetic microbeads</td><td>Miltenyi Biotec</td><td>130-049-801</td><td>Mix well prior use</td></tr><tr><td>Streptavidin microbeads</td><td>Miltenyi Biotec</td><td>130-048-101</td><td>Mix well prior use</td></tr><tr><td>Anti-Annexin V magnetic beads</td><td>Miltenyi Biotec</td><td>130-090-201</td><td>Mix well prior use</td></tr><tr><td>MACS LD&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-901</td><td></td></tr><tr><td>96-well U-bottom sterile culture plate</td><td>Greiner Bio-one</td><td>650180</td><td></td></tr><tr><td>96-well F-bottom sterile culture plate</td><td>Greiner Bio-one</td><td>655180</td><td></td></tr><tr><td>100 &amp;mu;m cell strainer mesh</td><td></td><td></td><td>To sterilize using UV radiation prior use</td></tr><tr><td>0.2 &amp;mu;m&amp;nbsp; sterile disposable filter units</td><td>Nalgene</td><td>567-0020</td><td>Can be substituted with any sterile filter device</td></tr><tr><td>CellTrace Violet</td><td>Invitrogen</td><td>C34557</td><td>CTV for short; alternative to CFSE</td></tr><tr><td>CellTrace Yellow</td><td>Invitrogen</td><td>C34567</td><td>CTY for short; alternative to CFSE</td></tr><tr><td>CellTrace Far Red</td><td>Invitrogen</td><td>C34564</td><td>CTFR for short; alternative to CFSE</td></tr><tr><td>Cell Proliferation Dye eFluor 670</td><td>eBioscience</td><td>65-0840</td><td>CPD670 for short; alternative to CFSE</td></tr><tr><td>PKH26</td><td>Sigma Aldrich</td><td>PKH26GL</td><td>PKH26, alternative to CFSE</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dextrose</td><td>Sigma Aldrich</td><td>G7021</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P5655</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma Aldrich</td><td>S5136</td><td></td></tr><tr><td>Phenol Red</td><td>Sigma Aldrich</td><td>P0290</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma Aldrich</td><td>C7902</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma Aldrich</td><td>P5405</td><td></td></tr><tr><td>Sodium chloride</td><td>Merck Millipore</td><td>S7653</td><td>Can use from other sources</td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma Aldrich</td><td>M2393</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma Aldrich</td><td>M2643</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma Aldrich</td><td>A9434</td><td></td></tr><tr><td>Tris-base</td><td></td><td></td><td></td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma Aldrich&amp;nbsp;</td><td>D8418</td><td></td></tr><tr><td>(5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)</td><td>Molecular Probes</td><td>C-1157</td><td>Reconstitute in DMSO</td></tr><tr><td>Phorbol 12,13-dibutyrate (PBDU, Phorbol ester)</td><td>Sigma Aldrich</td><td>P1269</td><td></td></tr><tr><td>A23187 (Calcium ionophore)</td><td>Sigma Aldrich</td><td>C7522</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies and recombinant protein&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD11b biotin (clone m1/70)</td><td>Biolegend</td><td>101204</td><td>T cell depletion cocktail</td></tr><tr><td>CD11c biotin (clone N418)</td><td>Biolegend</td><td>117304</td><td>T cell depletion cocktail</td></tr><tr><td>Gr-1 biotin (clone RB6-8C5)</td><td>Biolegend</td><td>108404</td><td>T cell depletion cocktail</td></tr><tr><td>Ter119 biotin (clone Ter119)</td><td>Biolegend</td><td>116204</td><td>T cell depletion cocktail</td></tr><tr><td>TCR-&amp;gamma;&amp;delta; biotin (clone GL-3)</td><td>Biolegend</td><td>118103</td><td>T cell depletion cocktail</td></tr><tr><td>CD19 biotin (clone 6D5)</td><td>Biolegend</td><td>115504</td><td>T cell depletion cocktail</td></tr><tr><td>B220 biotin (clone RA3-6B2)</td><td>Biolegend</td><td>103204</td><td>T cell depletion cocktail</td></tr><tr><td>CD49b biotin (clone DX5)</td><td>Biolegend</td><td>108904</td><td>T cell depletion cocktail</td></tr><tr><td>CD4 biotin (clone GK1.5)</td><td>Biolegend</td><td>100404</td><td>T cell depletion cocktail</td></tr><tr><td>CD8 biotin (clone 53-6.7)</td><td>Biolegend</td><td>100704</td><td>T cell depletion cocktail</td></tr><tr><td>F(ab&amp;rsquo;)2 goat anti-mouse IgM (plate coated)</td><td>Jackson ImmunoResearch&amp;nbsp;</td><td>115-006-075</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Anti-mouse CD40 mAb (plate coated)</td><td>Pharmingen&amp;nbsp;</td><td>553722</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Recombinant IL-4</td><td>ProSpec&amp;nbsp;</td><td>Cyt-282</td><td></td></tr><tr><td>LPS from E. coli Serotype 055:B5</td><td>Sigma Aldrich</td><td>L-4005</td><td></td></tr><tr><td>Anti-CD3 (clone clone OKT3) (plate coated)</td><td>eBioscience&amp;nbsp;</td><td>16-0037-85</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Anti-CD28 (clone clone 37.51) (plate coated)</td><td>eBioscience&amp;nbsp;</td><td>16-0281-85</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Recombinant IL-2</td><td>ProSpec</td><td>Cyt-370</td><td></td></tr><tr><td>Albumin from chicken egg white, Ovalbumin</td><td>Sigma Aldrich</td><td>A7641</td><td></td></tr></tbody></table>

isolation and activation of murine lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

depurazione cellule magnetica dei linfociti consente di purificare una popolazione di cellule bersaglio in un tempo relativamente breve lasso di tempo. Utilizzando il nostro protocollo di esaurimento, siamo stati in grado di aumentare la percentuale di cellule T CD8 (OT-I in ricombinazione attivante del gene-1 (RAG-1) topi -carente) dal 72,8% (prima della depurazione) al 94,2% (dopo la depurazione; Figura 1A) 4,5. Questi linfociti purificati possono poi essere...

Watch this Scientific Journal Video about Isolation and Activation of Murine Lymphocytes at JoVE.com

Isolation and Activation of Murine Lymphocytes

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

L&#39;isolamento e l&#39;attivazione di linfociti murini

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

isolation-and-activation-of-murine-lymphocytes

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

21.0K Views.  Nanyang Technological University. Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Video: Isolation and Activation of Murine Lymphocytes

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Modello murino di CD40-attivazione delle cellule B

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Ricerca

Immunologia e infezioni

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 ...

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Protocollo di isolamento del Mouse cellule dentritiche monocito-derivate e loro attivazione successiva In Vitro con tumore immunocomplessi

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

Ricerca sul cancro

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolamento di cellule T CD4 + da Linfonodi mouse utilizzando Miltenyi Purificazione MACS

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Biologia

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Isolamento dei linfociti da mouse mucosa del tratto genitale

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

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 ...

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 Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

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

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Isolando i linfociti del sistema immunitario intestinale piccolo per Mouse

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Isolamento delle cellule endoteliali dai capillari linfatici dermici murini

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Autore in primo piano: Isolamento basato su biglie magnetiche di cellule endoteliali linfatiche dermiche murine

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

Isolamento di cellule endoteliali linfatiche dermiche murine

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...

L'isolamento e l'attivazione di linfociti murini

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