Somos gratos a Drs. Markus Manz e Irving Weissman para a prestação de reagentes. Reconhecemos também o serviço prestado pela Citometria de Fluxo de Análise e Seleção celular Núcleo Facilidade da Primeira e Segunda laboratório central no National Taiwan University College of Medicine e NTU hospital, respectivamente. Este trabalho foi financiado pelo Ministério da Ciência e Tecnologia, Taiwan (MOST 102-2320-B-002-030-MY3) e os Institutos Nacionais de Investigação de Saúde, Taiwan (INDH-EX102-10256SI e INDH-EX103-10256SI).

Murganhos C57BL / 6 de tipo selvagem foram adquiridos a partir do Centro Nacional de Animais de Laboratório (NLAC), Taiwan. Todos os ratinhos foram criados e mantidos sob condições isentas de agentes patogénicos específicos. Protocolos e procedimentos de uso dos animais foram revisadas e aprovadas pelo cuidado e uso de animais Comitê Institucional da National Taiwan University College of Medicine (Permit Number: 20120075). Além disso, os pesquisadores fizeram todos os esforços para reduzir o potencial de dor, sofrimento, angústia ou nos animais durante a realização de experimentos. Todos os procedimentos descritos foram realizados à temperatura ambiente usando luvas. 1. Preparação de 6-ac células alimentadoras Nota: O AC-6,21 (AC-6 em curto) linha celular de estroma 18 (fornecida por I. Weissman, Universidade de Stanford) devem ser mantidas em RPMI suplementado com 15% de soro fetal de bovino inactivado pelo calor (FBS). Para servir como células de alimentação, AC-6-γ células são irradiadas com 3000 rad (30Gy) um dia antes da co-cultura para evitar a sua proliferação. Th notano AC-6 células tendem a perder a sua capacidade de suporte de diferenciação se deixou estar lotados durante a manutenção, ou se o seu número passagem é superior a 20. <ol><li> Lavar AC-6 células uma vez com 1 ml de solução salina tamponada com fosfato de Dulbecco (DPBS) e remover por aspiração DPBS. Tratar AC-6 células com 0,8 ml de solução de tripsina (tripsina a 0,05% e EDTA a 0,5 mM em DPBS) durante 3-5 min a 37 ° C, 5% de CO 2 e, em seguida, parar a reacção por diluição com 10 volumes de meio de cultura aos fazer a suspensão de célula única. </li><li> Semente AC-6 5.9x10 células em 4 células / poço em placas de 12 poços e incuba-se a 37 ° C, 5% de CO 2 O / N. Nota: A densidade da AC-6 células é muito importante, uma menor densidade favorecerá a geração CDC. Semear a uma densidade de 4 5.9x10 células / poço permitirão AC-6 para atingir confluência no dia seguinte, o que é óptimo para a derivação das PDC de CLPs. </li><li> Irradiar AC-6 células a 3000 rad (30 Gy), utilizando-γ irradiador. Nota: A dose utilizada para irradiar AC-6 é otimizado para evitar que as células se proliferem e ainda mantê-los viável a longo o suficiente para fornecer as citocinas e contato célula-célula necessárias para a diferenciação de DCs de CLPs. </li><li> Aspirar o meio e substituir com 1 ml de meio RPMI completo (RPMI com 10% de FBS inactivado pelo calor, 50 ug / ml de gentamicina e 50 pM β-mercaptoetanol). </li></ol> 2. Isolar BM As células de ratinhos <ol><li> Sacrifique ratos por asfixia com CO2 e deslocamento cervical. Colocar os ratinhos num tabuleiro de dissecação e esterilizar com etanol a 70%. Realizar uma incisão no abdómen e meados de remover a pele a partir da parte distal do rato, incluindo a pele que cobre as extremidades inferiores. </li><li> Dissecar ratinhos em uma capa semi-estéril. Para liberar o fêmur ea tíbia, o fêmur clipe acima e tíbias abaixo da articulação do joelho. Separar os músculos dos ossos usando uma tesoura e colocar os ossos numa placa de Petri 6 cm contendo 5 ml de RPMI completo. </li><li> Mover-se para uma capa de cultura estéril. Encha uma seringa de 3 ml ligada a uma agulha 27G com meio RPMI completo. Cortar ambas as extremidades dos ossos usando uma tesoura, e inserir a agulha 27G para liberar a medula de ossos, injetando suavemente RPMI completo uma vez a partir de cada extremidade. </li><li> Prepara-se uma suspensão de células individuais por pipetagem suave das células cima e para baixo 3-5 vezes na placa de cultura usando a seringa sem agulha. </li><li> Centrifugar as células durante 5 min a 500 xg e decanta-se o sobrenadante. </li><li> Lisar células vermelhas do sangue com 1 ml de tampão ACK (150 mM de NH4Cl, 10 mM KHCO 3, 0,1 mM de EDTA) a incubação durante 1 min e parar a reacção por diluição com 10 volumes de meio RPMI completo. Permitir que as células em repouso durante 5 min, de modo a sedimentar-se aglomerados de células mortas e detritos por gravidade tecido. Nota: Não fique mais de 5 min como isso irá resultar em perda de células devido à sedimentação de células viáveis. </li><li> Contendo células sobrenadante lentamente decante para um novo tubo deixando detritostrás no tubo inicial. </li><li> Centrifugar durante 5 minutos a 500 xg para sedimentar as células, em seguida, remover e descartar o sobrenadante. </li><li> Ressuspender as células totais BM (tipicamente 4-6 x 10 7 BM células são obtidas a partir de um murganho) em 100 ul de tampão FACS (1x PBS + 2% FBS + EDTA 1 mM). </li></ol> 3. FACS análise e classificação de CLPs <ol><li> Adicionar anti-CD16 / 32 (sobrenadante de hibridoma do clone 2.4G2, 50 ul / reacção ou 1-2 ug / reacção) em células em tampão de SCAF (passo 2.9), durante 1-2 minutos, a fim de bloquear os receptores de Fc. Nota: Anti-CD16 / 32 também é chamado bloco de Fc, que é adicionado para evitar ligação não específica dos anticorpos às células que expressam receptores de Fc, incluindo granulócitos, monócitos e linfócitos B. </li><li> Simultaneamente, as células são coradas com os seguintes anticorpos marcados com corantes fluorescentes (para 4x10 7 células) durante 15 min em gelo, evitando a luz ambiente. Antibodies: marcadores de linhagem PE-conjugados (0,2 ug de cada) incluindo o anti-CD3 (17-2), 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 ug de anti-c-Kit-PerCP-Cy5.5 (2B8), 1 ug de anti-Sca-1-FITC (D7), 0,4 ug de anti-M-CSFR-APC (AFS98), e 0,2 ug de anti-IL-7Rα-PE-Cy7 (A7R34). </li><li> Lavar as células com 3 ml de tampão de FACS, e centrifuga-se durante 5 min a 500 x g. </li><li> Volte a suspender as células em 300 ul de tampão de FACS e células filtrar através de um filtro de células 40 mM. </li><li> Lavar o tubo com um buffer adicional FACS 100 ul para recuperar quaisquer células restantes e filtrar para um FACS estéreis triagem tubo utilizando o mesmo filtro de células. </li><li> Realizar a análise de citometria de fluxo imediatamente após a coloração, usando filtros e voltagens adequadas para a detecção de sinal. Use laser de 488 nm para a detecção dos anticorpos com FITC, PerCP-Cy5.5-rotulados, do laser 561 nm, para detectar o PE- e anticorpos PE-Cy7-labelded e 633 nm laser de detectar o anticorpo marcado com APC. </li><li> Resolver CLPs de acordo com os seguintes marcadores de Lin - c-kit int int Sca-1 M-CSFR - IL-7Rα + (como coradas no passo 3.2), utilizando um classificador de células. Recolher as células separadas em um tubo de 15 ml contendo 8 ml de meio RPMI completo como uma almofada. </li><li> Grave o número absoluto de células classificadas como mostrado pelo classificador de células no final da corrida. Tipicamente, 5x10 4 CLPs podem ser obtidos a partir de MO de um rato. </li></ol> 4. co-cultura CLPs e AC-6 <ol><li> Centrifugar as células escolhidas a partir da etapa 3.7, durante 10 min a 500 xg, remover o sobrenadante e ressuspender o sedimento de células com meio RPMI completo suficientemente para se obter uma densidade celular de 5x10 4 em torno de células / ml. Semente 500 células / cavidade da placa de 12 poços contendo células alimentadoras, preparado no passo 1.4. </li><li> Adicionam-se 100 ng / ml FL 19 (Hu-Flt3L-Ig gerado internamente utilizando um sistema de expressão é fornecida por M. Manz, UniversiTy Hospital Zurique, Suíça) e incubar a 37 ° C, 5% de CO 2 com a monitorização visual periódica de desenvolvimento DC sob um microscópio. Nota: Comercialmente disponível FL recombinante humano ou rato FL pode ser utilizado como um substituto para a hu-Flt3L-Ig para suportar o desenvolvimento DC. </li><li> Recolher as células do sobrenadante ao dia 12 e lavar os poços uma vez com 0,5 ml de meio completo RPMI, combinando-se os sobrenadantes resultantes. Adicionar 0,5 ml de meio fresco e destruam as células aderentes com um raspador de células. </li><li> Combinar o meio contendo as células de ambas as partes e centrifugar durante 5 minutos a 500 x g. </li><li> Células sobrenadante e ressuspender em tampão FACS Decantar 50 ul. Adicionar 50 uL de anti-CD16 / 32 sobrenadante de hibridoma e incuba-se durante 1-2 minutos para bloquear os receptores Fc. </li><li> Enumerar e manchar todas as células com os seguintes anticorpos (0,05 ug) de cada anti-CD11c-APC (N418), anti-CD11b-FITC, e anti-B220-PE. </li><li> Lavagem e de centrifugação as células como descreverd no passo 3.3. </li><li> Volte a suspender as células em 100 ul de tampão FACS, portão CD11c + e analisar para CDCs (CD11c + CD11b + B220 -) e pDCs (CD11c + CD11b - B220 +) 17. </li></ol>

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Authors have nothing to disclose.

Aqui, descrevemos um sistema de cultura in vitro para a geração robusta de DCs, e pDCs em particular, a partir de um pequeno número de CLPs. A singularidade deste sistema de cultura é devido ao uso de AC-6, células de uma linha celular de estroma, como alimentadores. Esta abordagem tem sido mostrado para fornecer não só as citocinas, tais como IL-7, SCF, M-CSF e FL 20, mas também o contacto célula-célula 21, necessário para suportar o desenvolvimento DC. AC-6 células têm sido ...

As células dendríticas (CDs) são células apresentadoras de antigénios profissionais, que desempenham um papel importante no controlo da resposta imunitária 1. Enquanto as DCs são heterogéneos, eles podem ser classificados em duas populações, as DCs convencional (CDCs) e DCS plasmocitoides (PDCs) 2,3. Além de órgãos linfóides, CDCs pDCs e também são encontradas em tecidos incluindo o pulmão, intestino, pele e 2. A morfologia dos CDCs e pDCs difere, com CDCs expositoras projeções dendríticos-like e forma de pDCs ser como célula-mais de plasma. Além disso, o DC marcador rato comum, CD11c, é mais altamente expresso em CDCs que em pDCs. Além disso, os CDCs pode ainda ser dividido em CD11b + CD24 - CDCs e CD11b - CD24 + CDCs, sendo que ambos expressam níveis mais elevados de MHC classe II e de moléculas co-estimuladoras do que fazer pDCs 2. PDCs maduros, por outro lado, têm sido mostrados para expressar selectivamente Siglec-H e BST2 4. Functionally, CDCs são melhores células apresentadoras de antígeno que são pDCs; No entanto, pDCs pode produzir uma grande quantidade de interferão de tipo I sobre a infecção do vírus ou estimulação inflamatória 5. Ambos os CDCs e pDCs são de curta duração e, portanto, deve ser constantemente reabastecidos de progenitores dentro da medula óssea (BM) 6,7. A transferência adoptiva de células progenitoras comuns mielóides (PGZC) e progenitores linfóides comuns (CLPs) em ratinhos letalmente irradiados demonstra que CDCs e pDCs pode ser gerada a partir de ambas as linhagens 8-10. No entanto, há um subconjunto de pDCs que expressam RAG1 / 2 e possuem segmentos rearranjados DJ no locus de IgH 11,12. Estas células também partilhar semelhanças moleculares com linfócitos B, incluindo a expressão do marcador B220, receptores de detecção de ácidos nucleicos (TLR7 / TLR9), e factores de transcrição (e SPIB BCL11A) 13, dispõe de todos Acredita-se que as assinaturas de linhagem linfóide. Portanto, CLPs podem ser boas escolhas para in vitro geração de pDCs por causa da semelhança linhagem. Enquanto a frequência dos dois CDCs pDCs e em humanos e em ratos é muito baixo 6, DCs, particularmente CDC, podem ser geradas in vitro a partir de progenitores BM ou na presença de citocinas, tais como GM-CSF 11,14 ou ligando Flt3 (FL ) usando sistemas de livre-feeder 11,15,16. Infelizmente, no entanto, não é possível produzir um grande número de pDCs in vitro utilizando FL 11,15,16. Anteriormente tínhamos demonstrado que pDCs pode ser gerada de forma eficiente in vitro a partir CLPs que utilizam o sistema de alimentação de CA 6-17. A vantagem de utilizar a linha de células do estroma AC-6 no sistema de cultura é que ela proporciona um contacto célula-célula e a secreção de citoquinas que suportam a geração de grandes números de pDCs de CLPs. Embora a produção com este sistema é muito robusto, replicação cuidadosa dos procedimentos descritos a seguir é necessário iara garantir a geração eficiente de 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

Células dendríticas plasmocitï¿½des (PDCs) são poderoso tipo I interferon (IFN-a) que produzem células que são activados em resposta a infecção ou durante as respostas inflamatórias. Infelizmente, estudo da função PDC é prejudicada pela sua baixa freqüência em órgãos linfóides, e os métodos existentes para a geração in vitro DC predominantemente favorecer a produção de CDCs sobre pDCs. Aqui nós apresentamos uma abordagem única para gerar de forma eficiente pDCs de progenitores linfóides comuns (CLPs) in vitro. Especificamente, o protocolo descrito detalhes como purificar os CLPs de medula óssea e geram pDCs por coculturing com AC-6 células alimentadoras irradiadas com y na presença de ligando Flt3. Uma característica única do presente sistema de cultura é que as CLPs migrar sob o AC-6 e as células tornam-se as células formadoras de área de paralelepípedo, um passo crítico para a expansão pDCs. DCs morfologicamente distintas, a saber pDCs e CDCs, foram gerados após cerca de 2 semanas com uma composição de 70-90% pDCs em condições óptimas. Tipicamente, o número de pDCs geradas por este método é cerca de 100 vezes do número de CLPs semeadas. Portanto, este é um novo sistema com o qual gerar robustamente o grande número de pDCs necessárias para facilitar estudos adicionais sobre o desenvolvimento e a função destas células.

Um total de 7 4-6x10 células de MO são tipicamente isoladas a partir de fémures e tíbias de um 6-8 semanas de idade, de tipo selvagem C57BL / 6 mouse. Para resolver CLPs, células totais BM estão manchadas com anticorpos PE-conjugada contra marcadores de linhagem (CD3, CD8, B220, CD19, CD11b, GR-1, Thy1.1, NK1.1, Ter119, e MHC-II), anti -C-PerCP-Kit / Cy5.5, anti-Sca-1-FITC, anti-M-CSFR-APC e anti-IL-7Rα-PE / Cy7, analisadas e classificadas com um classificador de células. A estratégia de triagem para...

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 geração de células dendríticas plasmocitóides Murino de comum Linfóide Progenitores usando o sistema de alimentação AC-6

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 visualizações.  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. 

Vídeo: 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.

Geração e Rotulagem de murino derivadas da medula óssea Células Dendríticas com Nanocristais Qdot de Estudos de rastreamento

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

Imunologia e Infecção

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.

Isolar e imunocoloração linfócitos e células dendríticas a partir de placas de Peyer Murino

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.

Geração de uma nova vacina de células dendríticas usando melanoma e células-tronco de câncer escântico

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.

Avaliando o Desenvolvimento da murino células dendríticas plasmocitóides em placas de Peyer Usando transferência adoptiva de Progenitores hematopoiéticos

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.

Avaliando a inata Sensing de HIV-1 CD4 Infected<sup&gt; +</sup&gt; As células T por células dendríticas plasmocitóides Usando um<em&gt; Ex vivo</em&gt; Co-cultura do sistema.

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.

Geração de imatura, madura e tolerogênicas células dendríticas com diferentes fenótipos Metabolic

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.

Geração e identificação de GM-CSF macrófagos derivados e dendríticas células alveolares do tipo de ratinho de Medula Óssea

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.

Classificação de células activadas por fluorescência Para a purificação de células dendríticas plasmocitï¿½des do rato de Medula Óssea

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.

Geração de células dendríticas derivadas de monócitos humanos a partir de sangue inteiro

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.

Um novo sistema alimentador-livre para produção em massa de murino 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 ...