Siamo grati a Drs. Markus Manz e Irving Weissman per la fornitura di reagenti. Riconosciamo inoltre il servizio fornito dal citometria a flusso di analisi e cellulare ordinamento Nucleo Struttura del Primo e del Secondo Nucleo Laboratory presso la National Taiwan University College of Medicine e NTU ospedale, rispettivamente. Questo lavoro è stato sostenuto dal Ministero della Scienza e della Tecnologia, Taiwan (MOST 102-2320-B-002-030-MY3) e il National Institutes Health Research, Taiwan (NHRI-EX102-10256SI e NHRI-EX103-10256SI).

C57BL / 6 topi wild-type sono stati acquistati dal National Laboratory Animal Center (NLAC), Taiwan. Tutti i topi sono stati allevati e tenuti in condizioni esenti da organismi patogeni specifici. Protocolli e le procedure di utilizzo degli animali sono stati esaminati e approvati dal Comitato di cura e l&#39;uso degli animali istituzionale della National Taiwan University College of Medicine (Permesso Numero: 20.120.075). Inoltre, i ricercatori fatto ogni sforzo per ridurre il potenziale di dolore, la sofferenza, l&#39;angoscia negli animali durante l&#39;esecuzione di esperimenti. Tutte le procedure descritte sono state effettuate a temperatura ambiente con i guanti. 1. Preparazione di 6-AC cellule feeder Nota: L&#39;AC-6.21 (AC-6 in breve) linea di cellule stromali 18 (fornito da I. Weissman, della Stanford University) deve essere mantenuta in RPMI integrato con siero fetale bovino inattivato al calore del 15% (FBS). Per servire come cellule di alimentazione, AC-6 celle sono γ-irradiati con 3000 rad (30Gy) un giorno prima della co-coltura per evitare la loro proliferazione. Nota thAC-6 celle tendono a perdere la loro capacità differenziazione sostenere se lasciato sovraffollate durante la manutenzione, o se il loro numero passaggio è superiore a 20. <ol><li> Lavare AC-6 celle una volta con 1 ml di Dulbecco Phosphate-Buffered Saline (DPBS) e rimuovere DPBS per aspirazione. Trattare AC-6 cellule con 0,8 ml di soluzione di tripsina (0,05% tripsina e EDTA 0,5 mM in DPBS) per 3-5 minuti a 37 ° C, 5% CO 2 e quindi arrestare la reazione diluendo con 10 volumi di mezzo di coltura a fare sospensione singola cellula. </li><li> Seme AC-6 celle a 5.9x10 4 cellule / pozzetto in piastre da 12 pozzetti e incubare a 37 ° C, 5% di CO 2 O / N. Nota: La densità di AC-6 celle è molto importante in quanto una densità inferiore favorirà generazione CDC. Semina ad una densità di 5.9x10 4 cellule / pozzetto permetteranno AC-6 per raggiungere confluenza entro il giorno successivo, che è ottimale per la derivazione di pDCs da CLPS. </li><li> Irradiare AC-6 celle a 3000 rad (30 Gy) con γ-irradiatore. Nota: La dose utilizzata per irradiare AC-6 è ottimizzato per evitare che le cellule proliferazione e tuttavia mantenerli vitali sufficientemente lungo da assicurare le citochine e contatto cellula-cellula necessari per la differenziazione delle DC da CLPS. </li><li> Aspirare media e sostituirlo con 1 ml RPMI completo (RPMI con 10% di calore inattivato FBS, 50 mg / ml di gentamicina e 50 micron β-mercaptoetanolo). </li></ol> 2. Isolare BM cellule da topi <ol><li> Sacrifica topi di CO 2 asfissia e dislocazione cervicale. Posizionare i topi su un vassoio dissezione e sterilizzare con il 70% di etanolo. Fare un&#39;incisione a metà addome e rimuovere la pelle dalla parte distale del mouse compresa la pelle che copre le estremità inferiori. </li><li> Sezionare topi in una cappa semi-sterile. Per rilasciare il femore e tibia, femore agganciare sopra e tibie sotto del ginocchio. Staccare i muscoli dalle ossa con le forbici e posto le ossa in una capsula di Petri 6 centimetri contenente 5 ml RPMI completare. </li><li> Spostarsi in una cappa di coltura sterile. Riempire una siringa da 3 ml collegata ad un ago 27G con RPMI completo. Tagliare entrambe le estremità delle ossa con le forbici, e inserire l&#39;ago 27G per irrigare il midollo dalle ossa iniettando delicatamente RPMI completo una volta da ogni estremità. </li><li> Preparare una singola sospensione cellulare delicatamente pipettando le cellule su e giù per 3-5 volte in piatto cultura con la siringa senza ago. </li><li> Centrifugare le cellule per 5 minuti a 500 xg e decantare il surnatante. </li><li> Lisare globuli rossi con 1 ml di tampone di ACK (150 mm NH 4 Cl, 10 KHCO mm 3, 0,1 mM EDTA) incubazione per 1 min e fermare la reazione diluendo con 10 volumi di RPMI completo. Consentono alle cellule riposare per 5 min per pellet giù grumi di cellule morte e detriti di tessuto per gravità. Nota: non stare in piedi più di 5 min come questo si tradurrà in una perdita di cellule a causa della sedimentazione di cellule vitali. </li><li> Lentamente decantare il surnatante cellulare contenente un nuovo tubo lasciando detritidietro nel tubo originale. </li><li> Centrifugare per 5 minuti a 500 xg per far sedimentare le cellule, quindi rimuovere e scartare il surnatante. </li><li> Risospendere le cellule totali BM (tipicamente 4-6 x 10 7 cellule BM sono ottenuti da un topo) in 100 microlitri tampone FACS (PBS 1x + 2% FBS + 1 mM EDTA). </li></ol> 3. FACS Analisi e ordinamento di CLPS <ol><li> Aggiungi anti-CD16 / 32 (ibridoma supernant clone 2.4G2, 50 ml / reazione o 1-2 mg / reazione) in cellule in tampone FACS (passo 2.9) per 1-2 minuti, al fine di bloccare i recettori Fc. Nota: Anti-CD16 / 32 è anche chiamato blocco Fc, che si aggiunge a prevenire legame non specifico degli anticorpi di cellule esprimenti recettori Fc, tra cui i granulociti, monociti e linfociti B. </li><li> Contemporaneamente colorare le cellule con i seguenti anticorpi fluorescenti colorante marcati (per 4x10 7 cellule) per 15 min in ghiaccio, evitando luce ambientale. Anticorpi: marcatori lignaggio PE-coniugati (0,2 mcg ciascuna) tra cui anti-CD3 (17A2), anti-CD8 (53-6,7), anti-B220 (RA3-6B2), anti-CD19 (eBio1D3), anti-CD11b (M1 / 70), anti-Gr-1 (RB6-8C5), anti- Thy1.1 (HIS51), anti-NK1.1 (PK136), anti-TER119 (TER-119), e anti-MHC-II (NIMR-4), 0.2 mg anti-c-Kit-PerCP-Cy5.5 (2B8), 1 mg anti-Sca-1-FITC (D7), 0.4 mg anti-M-CSFR-APC (AFS98), e 0,2 mg anti-IL-7Rα-PE-Cy7 (A7R34). </li><li> Lavare le cellule con 3 ml di tampone FACS, e centrifugare per 5 min a 500 x g. </li><li> Risospendere le cellule in 300 microlitri di buffer FACS e celle filtranti attraverso un colino cella di 40 micron. </li><li> Lavare il tubo con un 100 microlitri di buffer FACS aggiuntivo per recuperare tutte le cellule residue e filtrare in un FACS sterile ordinamento tubo con la stessa colino cella. </li><li> Eseguire immediatamente citometria a flusso dopo la colorazione utilizzando filtri e tensioni appropriati per il rilevamento del segnale. Utilizzare 488 nm laser per rilevare gli anticorpi FITC, PerCP-Cy5.5-etichettati, 561 nm laser per rilevare il PE e PE-anticorpi Cy7-labelded e 633 nm laser per rilevare l&#39;anticorpo marcato APC. </li><li> Classificare CLPS secondo i seguenti marcatori lin - c-kit int Sca-1 int M-CSFR - IL-7Rα + (come macchiato al punto 3.2) utilizzando un cell sorter. Raccogliere le cellule ordinati in una provetta da 15 ml contenente 8 ml di RPMI completo come un cuscino. </li><li> Registrare il numero assoluto di cellule ordinati come mostrato dalla cell sorter alla fine della corsa. Tipicamente, 5x10 4 CLPS possono essere ottenuti dal BM di un topo. </li></ol> 4. coculture CLPS e AC-6 <ol><li> Centrifugare le cellule ordinati dal punto 3.7 per 10 minuti a 500 xg, rimuovere il surnatante e risospendere il pellet cellulare con abbastanza completo RPMI per ottenere una densità cellulare intorno 5x10 4 cellule / ml. Seed 500 cellule / pozzetto in 12-pozzetti contenenti cellule di alimentazione previste al punto 1.4. </li><li> Aggiungere 100 ng / ml FL 19 (hu-Flt3L-Ig generato internamente utilizzando un sistema di espressione fornita da M. Manz, University Hospital Zurigo, Svizzera) e incubare a 37 ° C, 5% CO 2 con controllo visivo periodico di sviluppo DC al microscopio. Nota: disponibile in commercio FL umana ricombinante o il mouse FL può essere utilizzato come un sostituto per hu-Flt3L-Ig per sostenere lo sviluppo continua. </li><li> Raccogliere le cellule nel supernatante a giorno 12 e lavare i pozzetti una volta con 0,5 ml di terreno RPMI completare combinando sovranatanti risultanti. Aggiungere 0,5 ml di mezzo fresco e rottami le cellule aderenti con un raschietto cellulare. </li><li> Combinare il mezzo cellulare contenente da entrambe le parti e centrifugare per 5 min a 500 x g. </li><li> Surnatante e risospendere le cellule decantare in 50 microlitri di buffer FACS. Aggiungere 50 microlitri anti-CD16 / 32 ibridomi surnatante e incubare per 1-2 minuti per bloccare i recettori Fc. </li><li> Enumerare e macchia tutte le celle con i seguenti anticorpi (0,05 mcg ciascuna) anti-CD11c-APC (N418), anti-CD11b-FITC e anti-B220-PE. </li><li> Lavare e centrifugare le cellule come descrivered al punto 3.3. </li><li> Risospendere le cellule in 100 microlitri di buffer FACS, cancello CD11c + e analizzare per CDC (CD11c + CD11b + B220 -) e pDCs (CD11c + CD11b - B220 +) 17. </li></ol>

<ol>
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Authors have nothing to disclose.

Qui si descrive un sistema in vitro in coltura per la robusta generazione di DC, e pDCs in particolare, da un piccolo numero di CLPS. L&#39;unicità di questo sistema di coltura è dovuto all&#39;uso di AC-6 cellule, una linea cellulare stromale, come alimentatori. Questo approccio ha dimostrato di fornire non solo le citochine, come IL-7, SCF, M-CSF e FL 20, ma anche il contatto cellula-cellula 21 necessario per sostenere lo sviluppo DC. AC-6 cellule sono state usate in precedenza per fac...

Le cellule dendritiche (DC) sono cellule presentanti l&#39;antigene professionale che svolgono un ruolo importante nel controllo risposte immunitarie 1. Mentre DC sono eterogenei, possono essere classificati in due popolazioni, DC convenzionale (CDC) e cellule dendritiche plasmacitoidi (PDC) 2,3. Oltre agli organi linfoidi, CDC e pDCs si trovano anche in tessuti tra cui polmone, intestino, pelle e 2. La morfologia di CDC e pDCs differenzia, con CDC espositrici proiezioni dendriti-simili e la forma di pDCs essere più plasma alveolare. Inoltre, il marcatore DC comune topo, CD11c, è più altamente espresso su CDCS che su pDC. Inoltre, CDC può essere ulteriormente suddivisa in CD11b + CD24 - CDC e CD11b - CD24 + CDC, entrambi i quali esprimono alti livelli di MHC di classe II e molecole costimolatorie di fare pDCs 2. PDC mature, d&#39;altra parte, hanno mostrato di esprimere selettivamente Siglec-H e BST2 4. Functionally, CDC sono antigene migliori presentano cellule rispetto sono pDCs; tuttavia, pDCs in grado di produrre una grande quantità di interferone di tipo I dopo l&#39;infezione da virus o la stimolazione infiammatoria 5. Entrambi i CDC e pDCs sono di breve durata, e di conseguenza, devono essere costantemente rifornite da progenitori all&#39;interno del midollo osseo (BM) 6,7. Trasferimento adottivo di progenitori comuni mieloidi (CMP) e progenitori linfoidi comuni (CLPS) in topi irradiati letalmente dimostra che i CDC e pDCs possono essere generati da entrambe le linee 8-10. Tuttavia, vi è un sottoinsieme di pDC che esprimono RAG1 / 2 e possiedono segmenti DJ riarrangiati al locus IgH 11,12. Queste cellule condividono anche analogie molecolari con linfociti B, tra cui l&#39;espressione del marcatore B220, recettori dell&#39;acido nucleico-sensing (TLR7 / TLR9), e fattori di trascrizione (SPIB e BCL11A) 13, dispone di tutto che si ritiene essere le firme del lignaggio linfoide. Pertanto, CLPs possono essere buone scelte per la generazione in vitro di pDCs causa di lignaggio somiglianza. Mentre la frequenza sia CDC e pDC negli esseri umani e nei topi è molto basso 6, DC, particolarmente CDC, può essere generato in vitro da BM o progenitori in presenza di citochine quali GM-CSF o 11,14 Flt3 ligand (FL ) utilizzando sistemi privi di alimentazione 11,15,16. Purtroppo, però, non è possibile produrre un gran numero di pDC in vitro su FL 11,15,16. In precedenza abbiamo dimostrato che pDCs possono essere efficacemente generati in vitro da CLPS utilizzando il sistema 17 AC-6 alimentatore. Il vantaggio di usare la linea cellulare stromale AC-6 nel sistema di coltura è che fornisce il contatto cellula-cellula e la secrezione di citochine che supportano la generazione di un gran numero di pDC da CLPS. Anche se la produzione con questo sistema è molto robusto, attento replica delle procedure descritte di seguito i è necessariol fine di garantire la generazione efficiente di pDCs.

<table>
	<tbody>
		<tr>
			<td>Anti-mouse Ly6g/Ly6c (PE), clone RB6-8C5</td>
			<td>Biolegend</td>
			<td>108408</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse NK1.1 (PE), clone PK136</td>
			<td>Biolegend</td>
			<td>108708</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse&#160; CD11b (PE), cloneM1/70</td>
			<td>Biolegend</td>
			<td>101208</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse CD19 (PE), clone eBio1D3</td>
			<td>Biolegend</td>
			<td>115508</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse B220 (PE), clone RA3-6B2</td>
			<td>Biolegend</td>
			<td>103208</td>
			<td>linage marker/FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse CD3 (PE), clone 17A2</td>
			<td>Biolegend</td>
			<td>100308</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse CD8a (PE), clone 53-6.7</td>
			<td>Biolegend</td>
			<td>100707</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse MHC-II (PE), clone NIMR-4</td>
			<td>Biolegend</td>
			<td>107608</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse Ter119 (PE), clone TER-119</td>
			<td>Biolegend</td>
			<td>116208</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse Thy1.1 (PE), clone HIS51</td>
			<td>eBioscience</td>
			<td>12-0900-83</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse M-CSFR (APC), clone AFS98</td>
			<td>Biolegend</td>
			<td>135510</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse c-Kit (PerCP/Cy5.5), clone 2B8</td>
			<td>Biolegend</td>
			<td>105824</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse Sca-1 (FITC), clone D7</td>
			<td>Biolegend</td>
			<td>108106</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse IL-7Ra (PE/Cy7), clone A7R34</td>
			<td>Biolegend</td>
			<td>135014</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11c (PerCP/Cy5.5), clone N418</td>
			<td>Biolegend</td>
			<td>117328</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b (FITC), clone M1/70</td>
			<td>Biolegend</td>
			<td>101206</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>FACSAria III</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Cell sorter</td>
		</tr>
		<tr>
			<td>FACS sorting tube&#160;</td>
			<td>BD Biosciences</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>FlowJo LLC</td>
			<td></td>
			<td>Flow analysis sofrware</td>
		</tr>
	</tbody>
</table>

in vitro generation of murine plasmacytoid dendritic cells from common lymphoid progenitors using the ac-6 feeder system

Cellule dendritiche plasmacitoidi (PDC) sono di tipo potente che interferone (IFN-I) che producono cellule che si attivano in risposta a infezioni o durante le risposte infiammatorie. Purtroppo, studio della funzione pDC è ostacolato dalla loro bassa frequenza in organi linfoidi, e metodi esistenti per la generazione vitro DC prevalentemente favoriscono la produzione di CDC sopra pDC. Qui vi presentiamo un approccio unico per generare efficientemente pDCs da progenitori linfoidi comuni (CLPS) in vitro. In particolare, il protocollo ha descritto i dettagli come purificare CLPS dal midollo osseo e generano pDCs da coculturing con AC-6 cellule di alimentazione gamma-irradiati in presenza di Flt3 ligando. Una caratteristica unica di questo sistema di coltura è che i CLPS migrano sotto le celle AC-6 e diventano cellule territoriali che formano acciottolate, un passaggio fondamentale per l&#39;espansione pDCs. DC morfologicamente distinte, vale a dire PDC e CDC, sono stati generati dopo circa due settimane con una composizione di 70-90% pDCs in condizioni ottimali. Tipicamente, il numero di pDC generati da questo metodo è circa 100 volte del numero di CLPS seminati. Pertanto, questo è un nuovo sistema con cui generare robusto il gran numero di pDC necessarie per agevolare ulteriori studi sviluppo e la funzione di queste cellule.

Un totale di 7 4-6x10 cellule BM sono in genere isolate da femori e tibie di un 6-8 sett-vecchio, wild-type C57BL / 6 del mouse. Per risolvere CLPS, cellule totali BM sono colorate con anticorpi coniugati PE contro marcatori lignaggio (CD3, CD8, B220, CD19, CD11b, Gr-1, Thy1.1, NK1.1, TER119, e MHC-II), anti -c-Kit-PerCP / Cy5.5, anti-Sca-1-FITC, anti-M-Cecoslovacchia-APC e anti-IL-7Rα-PE / Cy7, analizzati e ordinati con un cell sorter. La strategia di ordinamento per CLP è mostrato in Figu...

Watch this Scientific Journal Video about In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System at JoVE.com

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

In Vitro Generazione di Murine plasmacitoidi dendritiche Cellule dal comune linfoide Progenitori utilizzando l&#39;AC-6 Alimentatore Sistema

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

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Research

JoVE Journal

Immunology and Infection

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

8.3K Views.  National Taiwan University College of Medicine. In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand. 

Video: In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Generazione e l&#39;etichettatura di Murine osseo cellule dendritiche derivate dal midollo con nanocristalli Qdot per gli Studi inseguimento

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Ricerca

Immunologia e infezioni

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer&#039;s Patches

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Isolare e Immunostaining linfociti e le cellule dendritiche da placche di Peyer murino di

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

Generazione di un nuovo vaccino a cellule dendritiche con melanoma e cellule staminali tumorali squamose

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

Valutare lo sviluppo delle cellule murine plasmocitoidi dendritiche in placche del Peyer Utilizzando trasferimento adottivo di ematopoietiche Progenitori

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Valutare l&#39;innato rilevamento dell&#39;infezione da HIV-1 CD4 infetti<sup&gt; +</sup&gt; Cellule T dalle cellule dendritiche plasmacitoidi Utilizzando un<em&gt; Ex vivo</em&gt; Sistema di co-coltura.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Generazione di Immaturo, matura e tollerogenico dendritiche cellule con differenti fenotipi metabolica

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Generazione e identificazione di GM-CSF Derivato alveolo-come macrofagi e dendritiche cellule da midollo osseo di topo

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Separazione delle cellule fluorescenza-attivato per la purificazione delle cellule dendritiche plasmacitoidi dal midollo osseo di topo

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Generazione di derivate da monociti cellule dendritiche umane da sangue intero

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Un nuovo sistema privo di alimentatore per la produzione di massa di cellule di assassino naturali murini In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...