Estamos muy agradecidos a los Dres. Markus Manz y Irving Weissman para proporcionar reactivos. También reconocemos el servicio prestado por el análisis de citometría de flujo y Cell Sorting Core Instalación de la Primera y Segunda Laboratorio Core en la Universidad Nacional de Taiwán Facultad de Medicina y Hospital NTU, respectivamente. Este trabajo fue apoyado por el Ministerio de Ciencia y Tecnología, Taiwán (MOST 102-2320-B-002-030-MY3) y los Institutos Nacionales de Investigación de la Salud, Taiwán (INDH-EX102-10256SI y INDH-EX103-10256SI).

C57BL / 6 ratones de tipo salvaje se compraron desde el Centro Nacional de Animales de Laboratorio (NLAC), Taiwán. Todos los ratones fueron criados y mantenidos en condiciones libres de patógenos específicos. Protocolos y procedimientos de uso de animales fueron revisados ​​y aprobados por el Comité de Cuidado y Uso de Animales Institucional de la Universidad Nacional de Taiwán Colegio de Medicina (Número de Permiso: 20120075). Además, los investigadores hicieron todo lo posible para reducir el potencial de dolor, sufrimiento o angustia en los animales, mientras que la realización de experimentos. Todos los procedimientos descritos se llevaron a cabo a temperatura ambiente, mientras que el uso de guantes. 1. Preparación de 6-CA células Feeder Nota: El AC-6,21 (AC-6 en corto) línea celular estromal 18 (proporcionado por Weissman I., Universidad de Stanford) debe mantenerse en RPMI suplementado con suero inactivado por calor 15% bovino fetal (FBS). Para servir como células alimentadoras, AC-6-γ células son irradiadas con 3000 rad (30 Gy) un día antes de que el co-cultivo para evitar su proliferación. Nota ªAC-6 células tienden a perder su capacidad de diferenciación de apoyo si se deja de hacinamiento durante el mantenimiento, o si su número pasaje es más de 20. <ol><li> Wash AC-6 células una vez con 1 ml de fosfato solución salina tamponada de Dulbecco (DPBS) y quitar DPBS por aspiración. Tratar AC-6 células con 0,8 ml de solución de tripsina (0,05% de tripsina y EDTA 0,5 mM en DPBS) durante 3-5 min a 37 ° C, 5% de CO 2 y luego se detiene la reacción mediante la dilución con 10 volúmenes de medio de cultivo a hacer suspensión de células individuales. </li><li> Semilla AC-6 células en 5.9x10 4 células / pocillo en placas de 12 pocillos y se incuban a 37 ° C, 5% de CO 2 O / N. Nota: La densidad del AC-6 células es muy importante ya que una menor densidad favorecerá la generación de los CDC. La siembra a una densidad de 5.9x10 4 células / pocillo permitirán AC-6 para llegar a la confluencia al día siguiente, que es óptima para la derivación de PDC de CLPs. </li><li> Irradiar AC-6 células a 3000 rad (30 Gy) utilizando γ-irradiador. Nota: La dosis utilizada para irradiar AC-6 está optimizado para evitar que las células proliferen y, sin embargo mantenerlos viables tiempo suficiente para proporcionar las citoquinas y contacto célula-célula necesarios para la diferenciación de los países en desarrollo de CLPs. </li><li> Aspirar medio y reemplazar con 1 ml de RPMI completo (RPMI con inactivado por calor 10% de FBS, 50 mg / ml de gentamicina y 50 mM β-mercaptoetanol). </li></ol> 2. Las células Isolate BM a partir de ratones <ol><li> Sacrificar los ratones por asfixia con CO2 y dislocación cervical. Coloque los ratones en una bandeja de disección y esterilizar con etanol al 70%. Hacer una incisión en la mitad del abdomen y retirar la piel de la parte distal del ratón incluyendo la piel que cubre las extremidades inferiores. </li><li> Diseccionar los ratones en una campana semi-estéril. Para liberar el fémur y la tibia, cortar el fémur arriba y tibias por debajo de la articulación de la rodilla. Separar los músculos de los huesos utilizando tijeras y colocar los huesos en una placa de Petri de 6 cm que contiene 5 ml de RPMI completo. </li><li> Mover a una campana de cultivo estéril. Llene una jeringa de 3 ml conectada a una aguja 27G con RPMI completo. Cortar los dos extremos de los huesos con unas tijeras, e inserte la aguja 27G para limpiar la médula de los huesos inyectando suavemente RPMI completo una vez desde cada extremo. </li><li> Preparar una suspensión de células por pipeteo suave de las células arriba y abajo de 3-5 veces en la placa de cultivo utilizando la jeringa sin la aguja. </li><li> Centrifugar las células durante 5 min a 500 xg y decantar el sobrenadante. </li><li> Lisar células rojas de la sangre con 1 ml de tampón ACK (150 mM NH 4 Cl, 10 mM KHCO3, EDTA 0,1 mM) incubando durante 1 min y detener la reacción diluyendo con 10 volúmenes de RPMI completo. Permitir que las células en reposo durante 5 minutos con el fin de precipitar por grupos de células muertas y restos de tejido por la gravedad. Nota: No se pare más de 5 minutos ya que esto dará lugar a la pérdida de células debido a la sedimentación de células viables. </li><li> Lentamente decantar el sobrenadante que contiene células a un nuevo tubo de escombros dejandoatrás en tubo original. </li><li> Centrifugar durante 5 minutos a 500 xg para sedimentar las células, a continuación, retirar y desechar el sobrenadante. </li><li> Resuspender las células totales BM (típicamente 4-6 x 10 7 BM células se obtienen de un ratón) en 100 l de tampón FACS (PBS 1x + 2% de FBS + 1 mM EDTA). </li></ol> 3. FACS análisis y clasificación de CLPs <ol><li> Añadir anti-CD16 / 32 (clon de hibridoma sobrenadante 2.4G2, 50 l / reacción ó 1-2 g / reacción) en las células en tampón FACS (paso 2.9) durante 1-2 min con el fin de bloquear los receptores de Fc. Nota: Anti-CD16 / 32 también se denomina bloque de Fc, que se añade para evitar la unión no específica de anticuerpos a células que expresan receptores de Fc, incluyendo los granulocitos, monocitos y linfocitos B. </li><li> Simultáneamente teñir las células con los siguientes anticuerpos marcados con colorante fluorescentes (por 4x10 7 células) durante 15 min en hielo, evitando la luz ambiente. Anticuerpos: marcadores de linaje PE-conjugados (0,2 g cada uno) incluyendo 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), y anti-MHC-II (RMIN-4), 0,2 g anti-c-Kit-PerCP-Cy5.5 (2B8), 1 g anti-Sca-1-FITC (D7), 0,4 g anti-M-CSFR-APC (AFS98), y 0,2 g anti-IL-7Rα-PE-Cy7 (A7R34). </li><li> Lavar las células con 3 ml de tampón FACS, y centrifugar durante 5 min a 500 x g. </li><li> Resuspender las células en 300 L de tampón FACS y celdas de filtración a través de un filtro de células 40 M. </li><li> Lave el tubo con un tampón adicional 100 l FACS para recuperar las células restantes y filtrar en un tubo estéril FACS de clasificación utilizando el mismo filtro de células. </li><li> Realizar análisis de citometría de flujo inmediatamente después de la tinción utilizando filtros y tensiones adecuadas para la detección de señales. Utilice 488 nm láser para detectar los anticuerpos marcados con PerCP-Cy5.5 FITC,, 561 nm láser para detectar el PE y los anticuerpos PE-Cy7 labelded y 633 nm laser para detectar el anticuerpo marcado con APC. </li><li> Clasificar CLPs de acuerdo con los siguientes marcadores lin - c-kit int Sca-1 int M-CSFR - IL-7Rα + (como manchado en el paso 3.2) utilizando un clasificador de células. Recoger las células clasificadas en un tubo de 15 ml que contiene 8 ml de RPMI completo como un colchón. </li><li> Registre el número absoluto de células clasificadas como se muestra por el clasificador de células al final de la carrera. Típicamente, 5x10 4 CLPs se pueden obtener de la BM de un ratón. </li></ol> 4. cocultivo CLPs y AC-6 <ol><li> Centrifugar las células clasificadas desde el paso 3.7 durante 10 minutos a 500 x g, separar el sobrenadante y resuspender el botón celular suficiente RPMI completo para obtener una densidad celular alrededor de 5x10 4 células / ml. Seed 500 células / pocillo en la placa de 12 pocillos que contiene células alimentadoras preparados en el paso 1.4. </li><li> Añadir 100 ng / ml FL 19 (hu-Flt3L-Ig generado en la casa usando un sistema de expresión proporcionado por M. Manz, UniversiHospital de Ty Zürich, Suiza) y se incuba a 37 ° C, 5% de CO 2 con el monitoreo visual periódica del desarrollo DC bajo un microscopio. Nota: disponible comercialmente FL humana recombinante o el ratón FL se pueden utilizar como un sustituto para hu-Flt3L-Ig para apoyar el desarrollo DC. </li><li> Recoger las células en el sobrenadante a 12 días y lavar los pocillos una vez con 0,5 ml de medio RPMI completar la combinación de los sobrenadantes resultantes. Añadir 0,5 ml de medio fresco y desechar las células adherentes con un raspador de células. </li><li> Combinar el medio que contiene células de ambas partes y centrifugar durante 5 min a 500 x g. </li><li> Sobrenadante y resuspender las células en tampón FACS Decantar 50 l. Añadir 50 l anti-CD16 / 32 hibridoma sobrenadante y se incuba durante 1-2 minutos para bloquear los receptores Fc. </li><li> Enumerar y manchar todas las células con los siguientes anticuerpos (0,05 g cada uno) anti-CD11c-APC (N418), anti-CD11b-FITC y anti-B220-PE. </li><li> Lavar y centrifugar las células como describird en el paso 3.3. </li><li> Resuspender las células en 100 l de tampón FACS, puerta de CD11c + y analizar para el CDC (CD11c + CD11b + B220 -) y PDC (CD11c + CD11b - B220 +) 17. </li></ol>

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Authors have nothing to disclose.

Aquí se describe un sistema de cultivo in vitro para la generación de DCs robusta, y PDC, en particular, a partir de un pequeño número de CLPs. La singularidad de este sistema de cultivo se debe a la utilización de AC-6 células, una línea celular estromal, como alimentadores. Este enfoque ha sido demostrado para proporcionar no sólo las citoquinas, tales como IL-7, SCF, M-CSF y FL 20, sino también el contacto célula-célula 21 necesario para apoyar el desarrollo DC. AC-6 células...

Las células dendríticas (DC) son células presentadoras de antígeno profesionales que desempeñan un papel importante en el control de las respuestas inmunitarias 1. Mientras que los países en desarrollo son heterogéneos, que se pueden clasificar en dos poblaciones, convencional DC (CDC) y los DC plasmocitoides (PDC) 2,3. Además de los órganos linfoides, CDCs y PDC también se encuentran en tejidos incluyendo pulmón, intestino, piel y 2. La morfología de los CDC y PDC difiere, con CDC expositoras proyecciones dendríticas como y la forma de PDC son como células más plasma. Además, el marcador DC ratón común, CD11c, está más altamente expresado en CDC que en PDC. Por otra parte, CDC puede ser dividida en CD11b + CD24 - CDC y CD11b - CD24 + CDC, los cuales expresan mayores niveles de MHC de clase II y moléculas coestimuladoras que hacen PDC 2. PDC maduros, por otro lado, se ha demostrado para expresar selectivamente Siglec-H y BST2 4. Functionally, CDCs son mejores células presentadoras de antígenos que son PDC; sin embargo, PDC pueden producir una gran cantidad de interferón de tipo I sobre la infección por el virus o la estimulación inflamatoria 5. Ambos CDC y PDC son de corta duración, y por lo tanto, deben ser constantemente repuestos de progenitores dentro de la médula ósea (MO) 6,7. La transferencia adoptiva de células progenitoras mieloides comunes (CMPS) y progenitores linfoides comunes (CLPS) en ratones letalmente irradiados demuestra que CDCs y PDC pueden ser generados a partir de ambos linajes 8-10. Sin embargo, no es un subconjunto de PDC que expresan RAG1 / 2 y poseen segmentos DJ reordenados el locus IgH en 11,12. Estas células también comparten similitudes moleculares con linfocitos B, incluyendo la expresión del marcador B220, los receptores de ácido nucleico de detección (TLR7 / TLR9), y factores de transcripción (SPIB y BCL11A) 13, todas las características que se cree firmas del linaje linfoide. Por lo tanto, CLPs pueden ser buenas opciones para la generación in vitro de PDC, debido a la similitud linaje. Si bien la frecuencia de ambos CDCs y PDC en humanos y ratones es muy baja 6, países en desarrollo, en particular los CDC, se puede generar in vitro a partir BM o progenitores en presencia de citoquinas, tales como GM-CSF 11,14 o ligando Flt3 (FL ) utilizando sistemas de alimentación libre de 11,15,16. Desafortunadamente, sin embargo, no es posible producir un gran número de PDC in vitro utilizando FL 11,15,16. Anteriormente hemos demostrado que la PDC se pueden generar de manera eficiente in vitro a partir CLPs utilizando el sistema de alimentación AC-6 17. La ventaja de usar la línea celular estromal AC-6 en el sistema de cultivo es que proporciona contacto célula-célula y la secreción de citocinas que soportan la generación de un gran número de PDC de CLPs. Aunque la producción con este sistema es bastante robusto, i se requiere la replicación cuidadosa de los procedimientos descritos a continuaciónorden n para asegurar la generación eficiente de PDC.

<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 plasmacitoides (PDC) son potentes interferón de tipo I (células productoras de IFN-I) que se activan en respuesta a la infección o durante las respuestas inflamatorias. Por desgracia, el estudio de la función PDC se ve obstaculizada por su baja frecuencia en los órganos linfoides, y los métodos existentes para la generación in vitro DC favorecen predominantemente la producción de los CDC sobre PDC. Aquí presentamos un enfoque único para generar eficientemente PDC de progenitores linfoides comunes (CLPS) in vitro. Específicamente, el protocolo descrito detalles cómo purificar CLPs desde la médula ósea y generar PDC por cocultivo con AC-6 células alimentadoras gamma-irradiado en presencia de ligando de Flt3. Una característica única de este sistema de cultivo es que los CLPs migran por debajo de las células AC-6 y se convierten en células de adoquines en la zona de formación de, un paso crítico para la expansión de PDC. Morfológicamente distintos países en desarrollo, a saber, PDC y CDC, se generaron después de aproximadamente 2 semanas con una composición de 70-90% PDC en condiciones óptimas. Típicamente, el número de PDC generados por este método es más o menos 100 veces del número de CLPs sembradas. Por lo tanto, este es un sistema novedoso con el que para generar robustamente el gran número de PDC necesarias para facilitar estudios adicionales en el desarrollo y la función de estas células.

Un total de 4-6x10 7 células BM normalmente están aislados de los fémures y tibias de un solo 6/8 semanas de edad, de tipo salvaje C57BL / 6 mouse. Para resolver CLPs, células totales BM se tiñeron con anticuerpos conjugados con PE contra marcadores de linaje (CD3, CD8, B220, CD19, CD11b, Gr-1, Thy1.1, NK1.1, Ter119, y MHC-II), anti -c-Kit-PerCP / Cy5.5, anti-Sca-1-FITC, anti-M-CSFR-APC y anti-IL-7Rα-PE / Cy7, analizados y ordenados con un clasificador de células. La estrategia de clasificación de CLP...

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.

En Vitro Generación de células dendríticas murinas plasmocitoide de común linfoide Progenitores utilizando el sistema de alimentación AC-6

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

in-vitro-generation-murine-plasmacytoid-dendritic-cells-from-common

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 vistas.  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.

Generación y Etiquetado de murino médula ósea procedentes de células dendríticas con nanocristales Qdot de Estudios de seguimiento

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

Investigación

Inmunología e Infección

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.

Aislar y inmunoticción Los linfocitos y las células dendríticas de las placas de Peyer murinas de

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.

Generación de una nueva vacuna de células dendríticas usando melanoma y células madre escamosas del cáncer

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.

Evaluar el desarrollo de las células murino plasmocitoide dendríticas en las placas de Peyer Usando la transferencia adoptiva de Progenitores Hematopoyéticas

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.

La evaluación de la innata de detección de VIH-1 CD4 infectados<sup&gt; +</sup&gt; Células T por las células dendríticas plasmocitoides Usando un<em&gt; Ex vivo</em&gt; Co-cultura del 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.

Generación de inmaduras, maduras y tolerogénico células dendríticas con diferentes fenotipos metabólico

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.

Generación e identificación de GM-CSF Derivado alveolares-como los macrófagos y las células dendríticas A partir de médula ósea de ratones

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.

Clasificación de células activadas por fluorescencia para la purificación de células dendríticas plasmocitoide de la médula ósea de ratones

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.

Generación de células dendríticas derivadas de monocitos humanos de sangre entera

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 novedoso sistema de alimentador-libre para la producción masiva de células de asesino naturales murinos 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 ...