Este trabajo fue apoyado por el Fondo de Investigación Dotado Will y Jeanne Caldwell del Centro Oncológico Integral de la Universidad de Michigan y en parte por la subvención CA82529 de los NIH y la Fundación Gillson Longenbaugh.

1. Tinción de ALDEFLUOR
<ol><li>Preparación de muestras celulares: Prepare la suspensión unicelular de células tumorales, ya sea de células tumorales cultivadas o de muestras de tumor recién recolectadas. Cuente las células y ajuste la suspensión celular a una concentración de 1 x 106 células/ml en el tampón de ensayo ALDEFLUOR.</li>
	<li>Etiquete cuatro tubos de ensayo de poliestireno de 12 mm x 75 mm como tubos de control de la siguiente manera: #1: No manchados, #2: ALDEFLUOR, #3: ALDEFLUOR más DEAB, y #4: 7AAD.</li>
	<li>Para estos tubos de control, coloque la suspensión celular de 1 ml en #1 y #4, pero 2 ml en el tubo #2. Agregue 5 μl de DEAB (inhibidor de ALDH) en el tubo #3 y manténgalo en el hielo. A continuación, añadir 10 μl de sustrato ALDEFLUOR activado al tubo #2, mezclar y transferir inmediatamente 1 ml de la mezcla al tubo #3.</li>
	<li>Al mismo tiempo, añadir 2 μl de sustrato ALDEFLUOR activado por millón de células al resto de las células (tubos de muestra) también a 1 x 106 células/ml en el tampón de ensayo ALDEFLUOR.</li>
	<li>Incubar tanto los 4 tubos de control como los tubos de muestra durante 30 min en un baño de agua de 37 °C.</li>
	<li>Después de la incubación, agregue 5 μl 7AAD en el tubo de control #4, y 1 μl 7AAD 1 x 106 células al tubo de muestra. Incubar 10 min a 4 °C.</li>
	<li>Centrifugar todos los tubos durante 5 min a 250 x g y retirar el sobrenadante.</li>
	<li>Células resuspend en el buffer del ensayo de ALDEFLUOR.</li>
	<li>Realice la clasificación citología-basada del flujo.</li>
	<li>Establezca las puertas de clasificación utilizando células teñidas de ALDEFLUOR tratadas con DEAB como control negativo y las células teñidas de yoduro de propidio (PI) 7AAD- para el control de viabilidad. Sobre la base de estos controles, se estableció una puerta para distinguir las células ALDEFLUOR+/ALDHde alto,D5 y SCC7. Estas puertas se utilizarán para clasificar todas las células de muestra preparadas anteriormente para las células ALDEFLUOR+/ALDHaltas D5 y SCC7.</li>
</ol>2. Preparación de la vacuna de células madre de células madre de células cancerosas de células dendríticas de pulso (CSC-TPDC)
<ol><li>Eutanasia de ratones (singenéticos B6 y C3H, respectivamente) usando CO2 y aislar los huesos del fémur y la tibia.</li>
	<li>Ponga los huesos en etanol al 75% durante 1 min a temperatura ambiente. Luego lave los huesos usando HBSS.</li>
	<li>Corte la cabeza de los huesos y use una aguja de 21 G y una jeringa de 10 ml para empujar las células en un plato.</li>
	<li>Aspirar y soplar las células usando la jeringa para hacer la suspensión unicelular.</li>
	<li>Después de la centrifugación en 1.500 rpm durante 5 min, deseche el sobrenadante. A continuación, añadir 5 ml de tampón de lisis RBC a la lisis del RBC durante 1 min en un baño de agua de 37 °C.</li>
	<li>Cuente el número de células y ajuste la suspensión celular a una concentración de 1 millón de células/ml en medio completo (CM) que contenga 10 ng/ml GM-CSF y 10 ng/ml IL-4.</li>
	<li>Cultíe las células y compense el cm más IL-4 y GM-CSF 3 días más adelante.</li>
	<li>Al5º día, coseche las células dendríticas inmaduras (DC) y prepare el medio de aislamiento de CC que contiene 4,2 ml de solución C más 1 ml de medio de gradiente de densidad OptiPrep.</li>
	<li>Suspenda los pellets celulares en 5 ml CM. Añadir la suspensión celular en el medio de aislamiento lentamente. Centrífuga a 2.000 rpm a temperatura ambiente.</li>
	<li>Recoja los controladores de dominio entre el CM y el medio de aislamiento. Cuente el número de células y ajuste la suspensión celular a una concentración de 1 millón de células/ml en un medio de cultivo que contenga 10 ng/ml GM-CSF y 10 ng/ml IL-4.</li>
	<li>Prepare los lisatos tumorales suspendiendo las células D5 o SCC7altas o no clasificadas de ALDH en medio de cultivo y sujetas a exposiciones rápidas de congelación-descongelación cuatro veces seguidas de espín a ∼100 x g durante 5 min para recolectar la porción de membrana de los lisatos.</li>
	<li>Para preparar CSC-TPDC, pulse ADC con el lysate de célulasaltas autólogas de ALDH. Para preparar H-TPDC, pulse los CONTROLADORES DE DOMINIO con el lisato heterogéneo no clasificado de células tumorales. La proporción de DC a la célula tumorallysate es la misma.</li>
</ol>3. Vacunación y evaluación de la eficacia
<ol><li>Vacunar ratones B6 normales respectivamente con 2.500 células tumorales D5 CSC-TPDC o D5 H-TPDC por vía subcutánea(s.c.). Vacunar animales normales de C3H s.c. con 5.000 células tumorales SCC7 CSC-TPDC o SCC7 H-TPDC, respectivamente.</li>
	<li>Después de la vacunación, desafíe a los ratones B6 con las células heterogéneas del tumor D5 i.v. Euthanize los ratones usando elCO2 20 días más adelante. Cosechar los pulmones y enumerar las metástasis pulmonares.</li>
	<li>En el modelo SCC7, desafíe a los ratones C3H con células tumorales SCC7 s.c sin clasificar en el lado opuesto de la vacuna DC. Controle el tamaño del tumor.</li>
	<li>Al final de los experimentos eutanasia los ratones B6 y C3H utilizando CO2. En el día 34 después de la primera vacuna, eutanasia ratones con CO2,y al mismo tiempo recoger los bazos de cada grupo con un procedimiento aséptico.</li>
	<li>Active las células T y/o B del bazo con los mAb inmovilizados anti-CD3 más los mAbs anti-CD28 en el cm que contiene hrIL-2 o los LPS más las ascitis anti-CD40 (FGK45) del mAb.</li>
	<li>Después de la activación, recoger sobrenadantes y utilizar para el ensayo ELISA.</li>
	<li>Para el ensayo ELISA, recubre placas con anticuerpos para IFNγ, GM-CSF e IgG a 4 °C O/N.</li>
	<li>Después de la aplicación del tampón de bloqueo, agregue muestras y estándares e incube a temperatura ambiente.</li>
	<li>Lave la placa y agregue anticuerpos de detección de HRP y sustrato de TMB para la incubación.</li>
	<li>Mida la absorbancia en un lector de placas ELISA a 450 nm dentro de los 30 minutos después de detener la reacción.</li>
</ol>

<ol>
	<li>Shafee, N., Smith, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells+contribute+to+cisplatin+resistance+in+Brca1/p53-mediated+mouse+mammary+tumors.">Cancer stem cells contribute to cisplatin resistance in Brca1/p53-mediated mouse mammary tumors.</a> Cancer Res. 68, 3243-3250 (2008).</li><li>Nandi, S., Ulasov, I. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-dose+radiation+enhances+survivin-mediated+virotherapy+against+malignant+glioma+stem+cells.">Low-dose radiation enhances survivin-mediated virotherapy against malignant glioma stem cells.</a> Cancer Res. 68, 5778-5784 (2008).</li><li>Dallas, N. A., Xia, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoresistant+colorectal+cancer+cells,+the+cancer+stem+cell+phenotype,+and+increased+sensitivity+to+insulin-like+growth+factor-I+receptor+inhibition.">Chemoresistant colorectal cancer cells, the cancer stem cell phenotype, and increased sensitivity to insulin-like growth factor-I receptor inhibition.</a> Cancer Res. 69, 1951-1957 (2009).</li><li>Yamauchi, K., Yang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+cancer+metastasis+by+cyclophosphamide+pretreatment+of+host+mice:+an+opposite+effect+of+chemotherapy.">Induction of cancer metastasis by cyclophosphamide pretreatment of host mice: an opposite effect of chemotherapy.</a> Cancer Res. 68, 516-520 (2008).</li><li>Chang, A. E., Redman, B. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I+trial+of+tumor+lysate-pulsed+dendritic+cells+in+the+treatment+of+advanced+cancer.">A phase I trial of tumor lysate-pulsed dendritic cells in the treatment of advanced cancer.</a> Clin. Cancer Res. 8, 1021-1032 (2002).</li><li>Ito, F., Li, Q., Shreiner, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-CD137+monoclonal+antibody+administration+augments+the+antitumor+efficacy+of+dendritic+cell-based+vaccines.">Anti-CD137 monoclonal antibody administration augments the antitumor efficacy of dendritic cell-based vaccines.</a> Cancer Res. 64, 8411-8419 (2004).</li><li>Kirk, C. J., Hartigan-O'Connor, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell-dependent+antitumor+immunity+mediated+by+secondary+lymphoid+tissue+chemokine:+augmentation+of+dendritic+cell-based+immunotherapy.">T cell-dependent antitumor immunity mediated by secondary lymphoid tissue chemokine: augmentation of dendritic cell-based immunotherapy.</a> Cancer Res. 61, 2062-2070 (2001).</li><li>Ning, N., Pan, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cell+vaccination+confers+significant+antitumor+immunity.">Cancer stem cell vaccination confers significant antitumor immunity.</a> Cancer Res. 72, 1853-1864 (2012).</li><li>Ginestier, C., Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCR1+blockade+selectively+targets+human+breast+cancer+stem+cells+in+vitro+and+in+xenografts.">CXCR1 blockade selectively targets human breast cancer stem cells in vitro and in xenografts.</a> J. Clin. Invest. 120, 485-497 (2010).</li><li>Carpentino, J. E., Hynes, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aldehyde+dehydrogenase-expressing+colon+stem+cells+contribute+to+tumorigenesis+in+the+transition+from+colitis+to+cancer.">Aldehyde dehydrogenase-expressing colon stem cells contribute to tumorigenesis in the transition from colitis to cancer.</a> Cancer Res. 69, 8208-8215 (2009).</li><li>Charafe-Jauffret, E., Ginestier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+cell+lines+contain+functional+cancer+stem+cells+with+metastatic+capacity+and+a+distinct+molecular+signature.">Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature.</a> Cancer Res. 69, 1302-1313 (2009).</li></ol>

STEMCELL Technologies ha soportado las tarifas de publicación de Acceso Abierto.

Los huéspedes immunocompromised, tales como ratones de SCID, imposibilitar evaluaciones inmunológicas de CSCs debido a la carencia de la inmunidad adaptante dentro de los anfitriones. En este estudio, evaluamos el immunogenicity de CSCs en los anfitriones inmunocompetentes, que podrían mímico más de cerca ajustes pacientes. Los CSCs enriquecidos son inmunogénicos y podrían inducir una inmunidad protectora tumoral más efectiva cuando sus lisiados se cargan a los DCs como una vacuna en comparación con los CDC pulsados por lisiado de células tumorales no seleccionadas.  Mecánicamente, la protección fue conferida por la inducción selectiva de anticuerpos CSC-reactivos y de células T8 así como la producción de cytokine del tipo 1, e.g. IFNγ y GM-CSF.
La mayoría de las inmunoterapias actuales, incluidas las vacunas dendríticas basadas en células y la transferencia adoptiva de células T, están diseñadas para atacar antígenos diferenciados por tumores. CSCs, que puede no expresar estos antígenos distinguidos puede por lo tanto escapar de estas blancos inmunológicas. En cambio, la vacuna del CSC diseñada para apuntar específicamente a las células madres del cáncer puede destruir esta población especial de las células cancerosas, y mejorar así la eficacia terapéutica de la vacuna previniendo recaída y la metástasis del tumor.
En células cultivadas del tumor y tumores recientemente cosechados, identificamos a población CSC-enriquecida por el flujo cytometry basado en alta actividad de la deshidrogenasa del aldehído. Tales célulasaltas de ALDH podrían ser aisladas por la clasificación de flujo para ser utilizadas como una fuente de antígeno para pulsar DC para generar CSC-TPDC. Comparación utilizando célulasaltas de ALDH aisladas de células tumorales cultivadas vs de tumores recién cosechados no demostró ninguna diferencia significativa en el término de la inducción de la inmunidad anti-CSC8. Estos resultados revelaron el potencial para utilizar CSCs aislado de las células cultivadas del tumor o de los tumores recientemente cosechados para el uso clínico.
Para ser clínicamente relevante, una vacuna necesita ser examinada en un entorno terapéutico. Estos experimentos se están realizando ahora en nuestro laboratorio.

Las células madre cancerosas son relativamente resistentes a la quimioterapia y radioterapia convencionales1,2. Por otro lado, esta población de células podría ser la responsable de la recaída y progresión de los cánceres después de las terapias tradicionales contra el cáncer1-4. Debido a la falta de expresión de antígenos tumorales diferenciados en las células madre cancerosas, las células madre cancerosas pueden escapar de las intervenciones inmunológicas actuales de terapia para el cáncer, que están diseñadas principalmente para atacar los antígenos en las células tumorales diferenciadas. Por lo tanto, el desarrollo de nuevas estrategias dirigidas específicamente y destruyendo las células madre cancerosas puede tener promesas para aumentar la eficacia terapéutica del tratamiento actual del cáncer. Con este fin, se aislaron células madre cancerosas (CSC)-poblaciones enriquecidas a partir de dos tumores animales (melanoma D5 y cáncer de células escamosas SCC7), y los utilizamos como fuente de antígeno para pulsar las células presentadoras de antígeno (células dendríticas, DC) para preparar la vacuna CSC-TPDC. A continuación, se evaluó la inmunidad antitumoral inducida por la vacuna CSC-TPDC en los huéspedes inmunocompetentes singenéticos, ratones B6 y ratones C3H, respectivamente. La eficacia antitumoral inducida por CSC-TPDC se comparó con la vacuna tradicional de DC pulsada con linsato de células tumorales heterogéneas no clasificadas (H-TPDC), que ha sido utilizada previamente por nuestro grupo5,6,así como por otros investigadores7 tanto en estudios preclínicos como en ensayos clínicos.

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	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">ALDHEFLOUR KIT</td>
			<td style="width:399px;">Stemcell Technologies</td>
			<td style="width:219px;">1700</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Murine IL-4</td>
			<td style="width:399px;">Pepro Tech</td>
			<td style="width:219px;">214-14</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Murine GM-CSF</td>
			<td style="width:399px;">Pepro Tech</td>
			<td style="width:219px;">315-03</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Mouse IFN-&#611; ELISA KIT</td>
			<td style="width:399px;">BD Biosciences</td>
			<td style="width:219px;">555138</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Mouse GM-CSF ELISA KIT</td>
			<td style="width:399px;">BD Biosciences</td>
			<td style="width:219px;">555167</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">OptiPrep Density Gradient Medium</td>
			<td style="width:399px;">Sigma Aldrich</td>
			<td style="width:219px;">D1556</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">BD OptEIA TMB Substrated Reagent Set</td>
			<td style="width:399px;">BD Biosciences</td>
			<td style="width:219px;">555214</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;"></td>
			<td style="width:399px;"></td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Equipment</td>
			<td style="width:399px;"></td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">BD FACS Aria Cell Sorter</td>
			<td>BD Biosciences</td>
			<td>336834</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Kcjunior</td>
			<td style="width:399px;">Bio-Tek Instruments</td>
			<td style="width:219px;">176058</td>
		</tr>
	</tbody>
</table>

generation of a novel dendritic-cell vaccine using melanoma and squamous cancer stem cells

Identificamos la célula madre del cáncer (CSC) - poblaciones enriquecidas del melanoma murine D5 syngeneic a los ratones C57BL/6 y el cáncer squamous SCC7 syngeneic a los ratones de C3H usando ALDEFLUOR/ALDH como marcador, y probamos su inmunogenicidad usando el lysate de la célula como fuente de antígenos a las células dendríticas del pulso (DCs). Los DCs pulsados conlos altos lysates del CSC de ALDH indujeron inmunidad antitumores protectora perceptiblemente más alta que los DCs pulsados con los lysates de los lysates enteros unsorted de la célula del tumor en ambos modelos y en un ajuste de la metástasis del pulmón y un s.c. Ajuste del crecimiento del tumor, respectivamente. Este fenómeno era debido a las respuestas anti-CSC vacuna-inducidas y celulares del anti-CSC del CSC. En particular, los esplenocitos aislados del huésped sometido a la vacuna CSC-DC produjeron una cantidad significativamente mayor de IFNγ y GM-CSF que los esplenocitos aislados del huésped sometidos a la vacuna de lysate de células tumorales no clasificadas de CC pulsada. Estos resultados apoyan los esfuerzos para desarrollar una vacuna terapéutica CSC-basada autóloga para el uso clínico en un ajuste ayudante.

ALDEFLUOR/ALDH se ha utilizado como un solo marcador para aislar células madre en múltiples neoplasias malignas8-11. Identificamos poblaciones célula-enriquecidas madre del cáncer en dos modelos D5 y SCC7 del tumor usando ALDEFLUOR como marcador. Detectamos las células de ALDEFLUOR+ en el melanoma murine B16-D5 y el cáncer de célula squamous SCC7. Encontramos que las células ALDEFLUOR+ contribuyen aproximadamente 0,5% y 5,2% en líneas celulares tumorales D5 y SCC7 cultivadas, respectivamente (Figura 1). Las células tumorales recién cosechadas de tumores establecidos se han analizado para confirmar la existencia de células ALDEFLUOR+. Como también se muestra en la Figura 1,hubo 2,5% y 4,2% de las células ALDEFLUOR+ de tumores D5 y SCC7 establecidos in vivo, respectivamente. La tumorigenicidad y la capacidad de auto-renovación de estasaltas poblaciones clasificadas de D5 y SCC7 ALDEFLUOR+/ALDH fueron evaluadas en el huésped inmunocompetente singeneico, los ratones C57BL/6 y C3H, respectivamente8.
Se ha utilizado el lisato heterogéneo de células tumorales no clasificadas para pulsos de CDs (H-TPDC) tanto en estudios con animales como en ensayos clínicos5,6. Para examinar la inmunogenicidad de las CSCs, se aisló ALDHalto CSC y pulsado DC con el lisiado de las CSCs a CSC-TPDC generado(Figura 2)y se utilizó H-TPDC como un control convencional de la vacuna contra el cáncer para probar si CSC-TPDC tiene algún efecto beneficioso en la prevención del crecimiento tumoral.
Evaluamos la inmunogenicidad de CSCs examinando la inmunidad antitumores protectora inducida por CSC-TPDC. En el modelo D5, los ratones inmunocompetentes ingenuos fueron vacunados s.c con CSC-TPDC o H-TPDC (en el mismo lisato: cociente de LA C.C.). Los grupos control recibieron solución salina (PBS). Una semana después de la vacuna pasada, desafiaron a los ratones con las células sin clasificar del tumor D5 intravenoso (i.v.),y los pulmones fueron cosechados 3 semanas más adelante para enumerar metástasis del pulmón. Como se muestra en la Tabla 1,en comparación con el grupo PBS, los ratones tratados con el H-TPDC desarrollaron menos metástasis pulmonares. Es importante destacar que los ratones tratados con CSC-TPDC tenían significativamente menos metástasis pulmonares que el grupo de vacunas H-TPDC en ambos experimentos realizados. En el modelo SCC7, los animales normales de C3H fueron vacunados s.c con SCC7 CSC-TPDC o H-TPDC respectivamente en el flanco derecho, seguido por desafiando con las células sin clasificar s.c del tumor SCC7 en el flanco izquierdo. Comparado con el grupo de PBS, H-TPDC indujo inmunidad antitumores modesta contra crecimiento del tumor. Sin embargo, hubo una inhibición significativa del crecimiento tumoral en ratones que fueron tratados con CSC-TPDC en comparación con el grupo control y el grupo H-TPDC (p<0,05, Figura 3). Estos resultados indican que CSCs se podría utilizar como fuente más eficaz del antígeno para cargar DCs que las células sin clasificar tradicionales del tumor en la inducción de inmunidad protectora contra el desafío de las células del tumor.
Para entender más lejos los mecanismos que son la base de la inmunidad antitumores protectora CSC-inducida observada, cosechamos los bazos de los animales sujetados a las vacunas de la C.C. en el final de los experimentos. Las células del bazo entonces fueron activadas por anti-CD3/CD28/IL-2 o anti-CD3/CD28/IL-2 + LPS/anti-CD40. A continuación se recogieron los sobrenadantes de cultivo para detectar la expresión de citoquinas y anticuerpos.  Hubo producciones significativamente mayores de IFNγ y GM-CSF por parte de las células del bazo de los animales vacunados con D5 CSC-TPDC o SCC7 CSC-TPDC(Figura 4). Además, había perceptiblemente (p<0,05) una producción más alta de IgG por las células activadas LPS/anti-CD40 del bazo recogidas de los animales vacunados con D5 CSC-TPDC o SCC7 CSC-TPDC comparado con D5 H-TPDC o SCC7 H-TPDC. Estos anticuerpos fueron encontrados para atar a D5 y a SCC7 CSCs respectivamente, y tal atascamiento podría dar lugar a la lisis del CSC en presencia del complemento8.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig1highres.jpg" src="/files/ftp_upload/50561/50561fig1.jpg"> Figura 1. Lasaltas poblaciones de ALDHFLUOR+/ALDH fueron detectadas en tumores murine frescos cultivados así como nuevamente cosechados del melanoma D5 y de la célula squamous SCC7. Las células del tumor tratadas con 50 mmol/L DEAB, un inhibidor específico de ALDH, fueron utilizadas como el control negativo.
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig1highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a> 
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig2highres.jpg" src="/files/ftp_upload/50561/50561fig2.jpg"> Figura 2. Generación de vacunas de células madre cancerosas basadas en células dendríticas. Para la preparación de CSC-TPDC y de H-TPDC, las células dendríticas médula-derivadas fueron pulsadas con los lysatesaltos o unsorted de la célula del tumor de ALDH, respectivamente.
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig2highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a> 
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig3highres.jpg" src="/files/ftp_upload/50561/50561fig3.jpg"> Figura 3. La vacuna de DC pulsada con lysate CSC (CSC-TPDC) podría inducir una inmunidad antitumoral protectora más efectiva en el s.c. Modelo tumoral SCC7. <a href="https://www.jove.com/files/ftp_upload/50561/50561fig3highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a> 

<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig4highres.jpg" src="/files/ftp_upload/50561/50561fig4.jpg"> Figura 4. Respuestas celulares sistémicas más potentes en el huésped inmunocompetente vacunado con CSC-TPDC.  Los esplenocitos fueron cosechados de los animales sometidos a la vacunación H-TPDC o CSC-TPDC, y se activaron según lo indicado. Los sobrenadantes de la cultura entonces fueron recogidos para la detección del cytokine usando ELISA.
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig4highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a>

Watch this Scientific Journal Video about Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells at JoVE.com

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

Evaluada en huéspedes inmunocompetentes singenéticos, la vacuna de células dendríticas (DC) basada en células madre cancerosas (CSC) demostró una inmunidad antitumoral significativamente mayor que las vacunas tradicionales de DC pulsadas con células tumorales a granel heterogéneas.

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 ...

generation-novel-dendritic-cell-vaccine-using-melanoma-squamous

Research

JoVE Journal

Immunology and Infection

11.2K vistas.  University of Michigan. Evaluada en huéspedes inmunocompetentes singenéticos, la vacuna de células dendríticas (DC) basada en células madre cancerosas (CSC) demostró una inmunidad antitumoral significativamente mayor que las vacunas tradicionales de DC pulsadas con células tumorales a granel heterogéneas.

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

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

El análisis de la maduración pulmonar de células dendríticas y migración durante la inflamación de las vías alérgica

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Investigación

Inmunología e Infección

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

Análisis espectrométrico de masas de Antígenos glicoesfingolípido

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

La enumeración de las principales poblaciones de leucocitos de sangre periférica en ensayos clínicos multicéntricos utilizando un ensayo de sangre entera Fenotipado

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

De las Migraciones células dendríticas mediante resonancia 19F / 1H Magnetic

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

Generación de Arenavirus recombinante para el Desarrollo de Vacunas de la FDA aprobados células Vero

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

La identificación de mutaciones del ADN en Purificada hematopoyéticas células madre / progenitoras

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Usando Click Química para medir el efecto de la infección viral en el Host-Cell ARN Síntesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

Enriquecimiento de la célula automatizada de citomegalovirus células T específicas para aplicaciones clínicas utilizando el Sistema de citoquinas de captura

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

En Vivo Ensayo para la detección de la función citolítica de células T específicas de antígeno utilizando un modelo de vacunación

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Generación de un gran número de progenitores mieloides y células dendríticas precursores de médula Murine usando una célula nueva clasificación estrategia

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...