Reconocemos el flujo de USF Citometría Core Facility. Nos gustaría agradecer a la Dra. Denise Cooper para compartir recursos. También nos gustaría dar las gracias a Maya Cohen, Laura Pendleton y Latour de Diana por su ayuda en la creación y la filmación de este video. NN con el apoyo de la NSF FG-LSAMP puente hacia el desarrollo de recursos humanos de Becas de Doctorado # 0929435. Este trabajo fue financiado por la Sociedad Americana del Cáncer Institucional de Becas de Investigación # Centro del Cáncer 93-032-13/Moffitt otorgado a TG.

Antes de comenzar, preparar las siguientes soluciones: 3% de tinción Media (SM): -3% Suero bovino fetal (FBS) en 1X tampón fosfato salino (PBS) MACS búfer (MB): - 0,5% de albúmina de suero bovino (BSA) en 1XPBS 1. El bazo de los ratones de cosecha <ol><li> Inyectar por vía subcutánea de 6-8 semanas de edad C57BL / 6 ratones (Harlan), con 1,5 x 10 5 células murinas Panc02 en 100 l de PBS 1x (portador de tumor, la tuberculosis). Los ratones de control (ingenuo) recibirá 100 1XPBS l. </li><li> Aproximadamente 4 semanas después de la inyección, los ratones eutanasia por asfixia de dióxido de carbono. </li><li> El bazo de los ratones de la cosecha de disección roma con pinzas y tijeras a continuación, pesar con una balanza. Coloque en el bazo por separado, con la etiqueta 50 tubos cónicos que contienen 1 x PBS. </li></ol> 2. Generar un Suspensio una sola célulan de los leucocitos a partir de bazos Todos los procedimientos se debe realizar en un ambiente estéril bajo una campana de seguridad biológica y las células y los anticuerpos mantuvieron en hielo. <ol><li> Montar la disociación celular tamiz mediante la inserción de la pantalla de malla en la abertura de la copa hacia el fondo. Luego, inserte el anillo de retención en el área de la rosca con el lado ranurado y utilizar el llavero para apretar el anillo de retención, manteniendo así la pantalla en su lugar. Coloque el tamiz montado en una placa de Petri que contiene 10 ml de 1 x PBS. </li><li> Con bazos y maja vidrio uso para moler bazos contra la pantalla de malla de la disociación celular tamiz y en la placa de Petri. Repetir para cada grupo de tratamiento de los ratones. </li><li> Suspensión de células de filtro en un tubo cónico de 50 ml utilizando un colador 70 micras celular y una pipeta serológica de 5 ml. Centrifugar a 12.000 rpm (250-300 xg) durante 5 min. </li><li> Eliminar el sobrenadante y resuspender el sedimento en 5 ml de una solución amortiguadora de lisis de glóbulos rojos por el bazo x. Pipetear arriba y hacia abajo con fuerza. Incubar a temperatura ambiente durante 5 min. Parar la reacción añadiendo 20 ml de 1 x PBS. Pipetear arriba y hacia abajo con fuerza. Centrifugar a 12.000 rpm (250-300 xg) durante 5 min. </li><li> Eliminar el sobrenadante y resuspender el precipitado en 20 ml de PBS estéril 1 x. Pipetear arriba y hacia abajo con fuerza. </li><li> Contar células utilizando azul de tripano y un hemacitómetro y resuspende a una concentración deseada en el 3% SM de modo que 50 l es equivalente a 5x10 5-1x10 6 células (1x10 7 células / ml - 2x10 7 células / ml). </li></ol> 3. La tinción de la superficie celular / Inmunofenotipo de MDSC mediante citometría de flujo <ol><li> Etiquete los pocillos de una placa de fondo 96-V bien para el control y muestras experimentales y manchas individuales para los controles de compensación (no hay manchas, NS, Mac-1-FITC; Gr-1-APC y DAPI). </li><li> Añadir 5x10 5-1x10 6 células equivalentes a 50 l / pocillo de esplenocitos a sus respectivos pozos en el 96-V-así placa inferior. Centrifugarplaca a 12.000 rpm (250-300 xg) durante 5 min. </li><li> Preparar &quot;mezcla maestra&quot; (MM) de ratón BD Fc Bloque (rata anti-ratón CD16/32 anticuerpo monoclonal) diluido en 3% SM, en un tubo de microcentrífuga de 1,5 ml en hielo. Como punto de partida, utiliza 1 g Bloque Fc en 3% SM para un volumen final de 50 l, por pocillo. </li><li> Retirar con cuidado el sobrenadante de cada pocillo de la 96-V-así placa inferior mediante la rápida inversión de la placa sobre y hacia atrás, sobre un contenedor de desechos o sumidero sin interrupción de los sedimentos celulares. </li><li> Vortex, brevemente centrifugar MM Fc bloque durante 5 segundos y se añaden 50 l de todos los pellets en el 96-V-así placa inferior. Mezclar bien pipeteando suavemente hacia arriba y hacia abajo, dejando muestras en sus pozos. Incubar la placa durante 15 minutos en la oscuridad sobre el hielo. Centrifugar la placa a 12.000 rpm (250-300 xg) durante 5 min. </li><li> Preparar &quot;Master Mix&quot; (MM) de fluorocromo conjugado con anticuerpos diluidos en un 3% SM en un tubo de microcentrífuga de 1,5 ml en hielo, mientras que las muestras se incuban con el bloque de Fc. Los anticuerpos se debe ajustarpara determinar las diluciones óptimas para la tinción de los procedimientos. Como punto de partida, se combinan una dilución 1:25 de Mac-1-FITC y 1:20 dilución de Gr-1-APC en 3% SM para un volumen final de 50 l, por pocillo de muestra. </li><li> Retirar con cuidado el sobrenadante de cada pocillo de la 96-bien con fondo en V como se ha descrito anteriormente. </li><li> Vortex, brevemente centrifugar MM de anticuerpos conjugados con tinción fluorescente de 5 segundos y se añaden 50 l para el control y pellets de experimentación. Mezclar bien pipeteando suavemente hacia arriba y hacia abajo. Para una sola mancha compensaciones, añadir una dilución 1:25 de Mac-1-FITC, 1:20 dilución de Gr-1-APC y 75 ng / ml de DAPI, en 3% SM para un volumen final de 50 l por pocillo a sus respectivos pocillos. Añadir 50 l SM 3% de mancha así (no mancha). Mezclar bien e incubar las células en 96-y-V placa inferior durante 30 minutos en la oscuridad en hielo. </li><li> FACS Label tubos (5 ml, 12 mm x 75 mm de poliestireno tubos de fondo redondo) que corresponden a cada pocillo en la placa de 96 pocillos con fondo en V. Añadir 200 l SM 3% a cada uno de FACStubo. </li><li> Centrifugar la placa a 12.000 rpm (250-300 xg) durante 5 min y eliminar sobrenadantes. Lavar gránulos mediante la adición de 100 l SM 3% a cada sedimento y mezclar bien pipeteando suavemente hacia arriba y hacia abajo. Centrifugar la placa a 12.000 rpm (250-300 xg) durante 5 min. Repita el paso de lavado, una vez más. </li><li> Retirar con cuidado el sobrenadante de cada pocillo de la 96-bien con fondo en V como se ha descrito anteriormente. Resuspender cada sedimento en 100 l SM 3% y mezclar bien. </li><li> Transferir 100 l precipitado resuspendido de cada pocillo de la 96-bien con fondo en V a su placa de tubo, respectivamente, etiquetados FACS. </li><li> Antes de análisis de flujo de la citometría, añadir 75 ng / ml DAPI para el control y muestras experimentales y de control de DAPI sola mancha de compensación. </li><li> Realizar la adquisición de citometría de flujo de datos de los porcentajes de MDSC. Lleve a cabo una compensación con el negativo (sin control de mancha) y los controles positivos individuales. Crear un gráfico de puntos que muestra la dispersión frontal (FSC) versus lateral (SSC) en escala logarítmica de forma que las poblaciones de leucocitos deinterés pueden ser identificados. Dibuja una puerta grande en todos los leucocitos, con exclusión de los desechos y grupos con menor avance y la dispersión lateral. Desde esta puerta principal, cree un gráfico de puntos que muestra nueva cooperación Sur-Sur en comparación con DAPI y la puerta de DAPI (en vivo) de las células. Seleccione esta población recién cerrada y crear un gráfico de puntos que muestra Mac-1 en comparación con Gr. 1-y la puerta de su doble positivo (Mac-1 + Gr-1 +) MDSC. </li></ol> 4. Enriquecimiento magnética de Gr-1 + leucocitos <ol><li> Alícuotas 1x10 7 restantes leucocitos teñidas en tubos debidamente etiquetados FACS y centrifugar a 12.000 rpm (250-300 xg) durante 5 min. </li><li> Preparar MM de Gr-1-PE anticuerpo en un tubo de microcentrífuga de 1,5 ml. Para un máximo de 10 7 células, utilizar una dilución 1:10 de Gr-1-PE anticuerpo en 50 MB l, por muestra. Por el número de células mayores, aumentar el volumen en consecuencia. En pocas palabras centrifugar durante 5 segundos, se suman a los leucocitos en los tubos de FACS y se incuba durante 15 min a 4 ° C en la oscuridad. </li&gt; <li> Añadir 2 ml de MB a los tubos de FACS, se centrifuga a 12.000 rpm (250-300 xg) durante 5 minutos y desechar el sobrenadante. </li><li> Preparar MM de Anti-PE MicroBeads en un tubo de microcentrífuga de 1,5 ml. Para un máximo de 10 7 células, use una dilución 1:4 de anti-PE MicroBeads en 200 MB l, por ejemplo. Por el número de células mayores, aumentar el volumen en consecuencia. En pocas palabras centrifugar durante 5 segundos, se suman a los leucocitos en los tubos de FACS y se incuba durante 15 min a 4 ° C en la oscuridad. </li><li> Añadir 2 ml de MB a los tubos de FACS, se centrifuga a 12.000 rpm (250-300 xg) durante 5 minutos y desechar el sobrenadante. Resuspender el sedimento en 3 ml de SB. Filtrar a través de un colador 70 micras en un nuevo tubo, con la etiqueta 50 ml cónico. </li><li> Preparar y el primer separador MACS Auto Pro. Recarga todas las botellas con las soluciones adecuadas y vaciar la botella de residuos, si es necesario. Encienda el instrumento y examinar el estado de los depósitos de fluidos y de la columna (s) después de la inicialización. Todos los símbolos deben ser de color verde. En el menú, seleccione &quot;separación&quot; de la parte superiorbarra de menú, seguido de &quot;Lavar ahora&quot; en la barra de menú inferior. Seleccione &quot;Rinse&quot; de la opción de pop-up, seguido de &quot;Ejecutar&quot; para iniciar el proceso de cebado. Una vez que el proceso de cebado se completa con éxito, el instrumento mostrará entonces que es &quot;Listo para la Separación&quot; en el menú de estado. </li><li> Elija bastidor adecuada del tubo fría para tamaños de tubo y el lugar 50 ml tubo cónico con células marcadas magnéticamente en la fila A, 50 ml tubo cónico para la recogida de fracción negativa en la línea B y 15 ml tubo cónico para la recogida de la fracción positiva en la fila C. </li><li> Seleccione la opción &quot;POSSEL_S&quot; programa de separación de células para la selección positiva de las células diana marcadas en el modo de sensibilidad de las muestras. Magnéticamente células diana marcadas son retenidos en la columna de autómatas; células no marcadas se liberan en el tubo de recogida de fracción negativa en B. En la fila retracción automática del imán, las células diana marcadas se libera en el tubo de recogida positivo en la fila C del tubo bastidor. </li></ol>+ leucocitos enriquecidos <ol><li> Recuento de Gr-1 + y Gr-1 - Fracciones con azul de tripano y un hemacitómetro. Resuspender las células en la concentración deseada en el medio de la tinción de 3% en lo que el 50 l es equivalente a 5x10 5-1x10 6 células (1x10 7 células / ml - 2x10 7 células / ml) y transferir 50 l de tubos etiquetados correspondientemente FACS. </li><li> Preparar un MM de Mac-1 solamente, a una dilución 1:25 en 3% SM para un volumen final de 50 l por muestra. Teñir las células individuales y preparar las compensaciones mancha de Gr-PE, Mac-1 FITC y DAPI para el análisis de citometría de flujo. Añadir 200 l 3% SM y colorante DAPI viabilidad como se ha descrito anteriormente. </li><li> Realizar la adquisición de citometría de flujo de datos para determinar Gr-1 + y Gr-1 - porcentajes y también para comparar porcentajes MDSC enriquecimiento de pre-y post-autoMACS. </li></ol> 6. Los resultados representativos Aquí se muestra r<html> epresentante resultados para el enriquecimiento autoMACS de Gr-1 + leucocitos de agrupados, el bazo ingenuos para FACS posteriores de clasificación de la MDSC (Figura 1). 1x10 6 leucocitos ingenuos fueron teñidas con Mac-1-FITC y anticuerpos Gr-1-APC para identificar los porcentajes MDSC utilizando un instrumento BD LSRII, antes de la clasificación autoMACS. 1x10 7 leucocitos ingenuos fueron teñidas con anti-Gr-1-PE anticuerpos y de educación física de microesferas para el enriquecimiento de Gr-1 + leucocitos mediante un separador de autoMACS Pro. Post-autoMACS enriquecimiento, Gr-1 los porcentajes de Gr-1 + y Gr-1 - fracciones recogidas fueron evaluadas mediante citometría de flujo. 1x10 6 Gr-1 + leucocitos fueron removidos y se tiñeron con Mac-1-FITC para analizar y comparar los porcentajes de MDSC de enriquecimiento, se agruparon los leucocitos ingenuos que no enriquecido, se reunieron con tumores leucocitos por citometría de flujo. fig1.jpg &quot;/&gt; Figura 1. AutoMACS enriquecimiento de Naïve Gr-1 + leucocitos para FACS MDSC Clasificación. Los bazos se recogerá a partir de los ratones portadores de tumores de páncreas e ingenuo y se transforma en una sola célula suspensiones. El análisis de citometría de flujo de la superficie de los leucocitos ingenuo tiñeron con Mac-1 y Gr-1 anticuerpos fluorescentes conjugados, antes de enriquecimiento autoMACS (A). Análisis de flujo de la citometría de Gr-1 + (B) y Gr-1 - (C) las fracciones de post-autoMACS enriquecimiento de Gr-1 + en las células de los agrupados leuckocytes ingenuos manchadas de Gr-1-PE anticuerpos y microesferas de anti-PE. Análisis de flujo de la citometría de MDSC y Gr-1 + porcentajes post-autoMACS enriquecimiento de Gr-1 + en las células de leucocitos agrupados ingenuos (D) en comparación con los no enriquecidos leucocitos agrupados de ratones portadores de tumores (E) (los ratones no tratados previamente, n = 5 ; ratones portadores de tumores, n = 3). MDSC y los porcentajes de Gr-1 es cerrada en las parcelas representativas de contorno y los histogramas.

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Este es un método detallado para el procesamiento y immunophentyping poblaciones MDSC que es aplicable a diferentes tejidos linfoides de varios modelos animales. En particular, el enriquecimiento autoMACS puede ser utilizado para el aislamiento de varias poblaciones de leucocitos, incluyendo Gr-1 agotamiento de esplenocitos 4, la purificación de subconjuntos mieloides de esplenocitos y ganglios linfáticos 5, el aislamiento de los neutrófilos de médula ósea 14 y purificación de cé...

<table border="1"><tbody><tr><td> REACTIVO </td><td> EMPRESA </td><td> Catálogo # </td><td> COMENTARIOS </td></tr><tr><td> Fosfato 1X solución salina tamponada </td><td> Thermo Scientific Hyclone </td><td> SH30028.02 </td><td> Ca 2 + / Mg 2 + / rojo fenol libre </td></tr><tr><td> La albúmina de suero bovino (BSA) </td><td> Sigma-Aldrich </td><td> A7906 </td><td> Vamos a BSA se disuelven sin problemas en PBS, medio ambiente estéril </td></tr><tr><td> Suero fetal bovino (SFB) </td><td> Thermo Scientific Hyclone </td><td> SV3001403HI </td><td> Inactivadas por calor, ambiente estéril </td></tr><tr><td> Rata anti-ratón CD16/32 anticuerpo monoclonal (Fc Bloque) </td><td> BD Biosciences </td><td> 553142 </td><td> Ambiente estéril </td></tr><tr><td> Anti-ratón CD11b (Mac-1) FITC </td><td> eBiosciences </td><td> 11-0112 </td><td> EstérilMedio ambiente </td></tr><tr><td> Anti-ratón Ly6G (Gr-1) de APC </td><td> eBiosciences </td><td> 17-5931 </td><td> Ambiente estéril </td></tr><tr><td> Anti-ratón Ly6G (Gr-1) PE </td><td> eBiosciences </td><td> 12-5931 </td><td> Ambiente estéril </td></tr><tr><td> DAPI </td><td> Invitrogen </td><td> D1306 </td><td> Medio Ambiente de dilución estéril de serie </td></tr><tr><td> Disociación celular Tamiz </td><td> Sigma-Aldrich </td><td> CD1-1KT </td><td> Autoclave antes de su uso </td></tr><tr><td> 70-m colador </td><td> BD Biosciences </td><td> 352350 </td><td> Ambiente estéril </td></tr><tr><td> 1X tampón de lisis de glóbulos rojos </td><td> eBiosciences </td><td> 00-4333-57 </td><td> Calentar a temperatura ambiente antes de su uso; ambiente estéril </td></tr><tr><td> Placas de Petri </td><td> Fisher Scientific </td><td> 08-757-12 </td><td> Ambiente estéril </td></tr><tr><td> Con 50 mltubos de cos </td><td> Thermo Scientific </td><td> 339652 </td><td> Ambiente estéril </td></tr><tr><td> 5ml 12X75mm poliestireno tubos de fondo redondo </td><td> BD Biosciences </td><td> 352054 </td><td> Ambiente estéril, conocidos como tubos FACS </td></tr><tr><td> 96 pocillos fondo en V placas </td><td> Corning </td><td> 3897 </td><td> Ambiente estéril </td></tr><tr><td> Azul de tripano </td><td> Cellgro </td><td> 25-900-CI </td><td> Ambiente estéril </td></tr><tr><td> PE MicroBeads </td><td> Miltenyi Biotec </td><td> 130-048-801 </td><td> Ambiente estéril </td></tr><tr><td> AutoMACS Pro Separador </td><td> Miltenyi Biotec </td><td> 130-092-545 </td><td></td></tr><tr><td> AutoMACS Columnas </td><td> Miltenyi Biotec </td><td> 130-021-101 </td><td></td></tr><tr><td> AutoMACS funcionamiento de amortiguación </td><td> Miltenyi Biotec </td><td> 130-091-221 </td><td&gt; </td></tr></tbody></table>

preparation of myeloid derived suppressor cells (mdsc) from naive and pancreatic tumor-bearing mice using flow cytometry and automated magnetic activated cell sorting (automacs)

MDSC son una población heterogénea de los macrófagos, células dendríticas inmaduras y granulocitos que se acumulan en los órganos linfoides en condiciones patológicas, incluyendo la infección parasitaria, inflamación, estrés postraumático, la enfermedad de injerto contra huésped, la diabetes y el cáncer de 1-7. En ratones, MDSC expresa Mac-1 (CD11b) y Gr-1 (Ly6G y Ly6C) antígenos de superficie 7. Es importante señalar que MDSC se ha estudiado bien en varios portadores de tumores anfitriones donde son significativamente expandidas y reprimir anti-tumorales respuestas inmunes frente a las contrapartes no tratados previamente 7-10. Sin embargo, dependiendo de la condición patológica, hay diferentes subpoblaciones de MDSC con distintos mecanismos y objetivos de supresión 11,12. Por lo tanto, los métodos eficaces para aislar poblaciones viables MDSC son importantes para dilucidar sus diferentes mecanismos moleculares de supresión in vitro e in vivo. Recientemente, el GHansah grupo ha informado de la expansión de MDSC en un modelo murino de cáncer de páncreas. El portador de tumor MDSC muestran una pérdida de la homeostasis y el aumento de la función supresora en comparación con el ingenuo MDSC 13. MDSC porcentajes son significativamente menores en los compartimentos linfoides de los ratones no tratados frente a un tumor-cojinete. Esta es una advertencia importante, que a menudo dificulta la precisión de estos análisis comparativos MDSC. Por lo tanto, enriquecer la Gr-1 + leucocitos de ratones no tratados previamente antes de la clasificación celular activado por fluorescencia (FACS) mejora la pureza, la viabilidad y reduce significativamente el tiempo de ordenación. Sin embargo, el enriquecimiento de Gr-1 + leucocitos de ratones portadores de tumores es opcional ya que se encuentran en abundancia para la FACS rápidas de clasificación. Por lo tanto, en este protocolo, se describe un método muy eficaz de inmunofenotipo MDSC y enriquecer Gr-1 + leucocitos del bazo de los ratones no tratados previamente para ordenar MDSC de una manera oportuna. Ratones inmunocompetentes C57BL / 6 se inoculan con murino ce Panc02LLS por vía subcutánea, mientras que los ratones no tratados previamente recibirá 1XPBS. Aproximadamente 30 días después de la inoculación, el bazo se cosechan y se procesan en una sola célula suspensiones utilizando una celda de la disociación tamiz. A continuación se esplenocitos de glóbulos rojos (RBC) se lisaron y una alícuota de estos leucocitos se tiñen con un fluorocromo conjugado con anticuerpos contra la Mac-1 y Gr-1 a los porcentajes MDSC inmunofenotipo por citometría de flujo. En un experimento paralelo, los leucocitos de los ratones no tratados enteros están manchadas con fluorescentes conjugados con 1 gr de anticuerpos, se incubaron con el PE-MicroBeads y seleccionados de manera positiva con un sistema automatizado magnético de células activadas clasificación (autoMACS) Pro Separator. A continuación, una parte alícuota de Gr-1 + leucocitos se tiñen con Mac-1 anticuerpos para identificar el aumento en porcentajes MDSC utilizando citometría de flujo. Ahora bien, estos Gr1 + leucocitos enriquecidos están listos para la clasificación de MDSC FACS para ser utilizados en los análisis comparativos (ingenuo vs tumor cojinete-) in vivo e in vitro </em&gt; ensayos.

Watch this Scientific Journal Video about Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell S

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

Este es un método rápido y completo de inmunofenotipo mieloide derivados de células supresoras (MDSC) y enriquecedora Gr-1<sup&gt; +</sup&gt; Leucocitos de bazos de ratones. Este método utiliza la citometría de flujo y Celular AutoMACS clasificación para enriquecer viables Gr-1<sup&gt; +</sup&gt; Leucocitos antes de FACS clasificación de MDSC para su uso<em&gt; En vivo</em&gt; Y<em&gt; In vitro</em&gt; Ensayos.

Preparación de células mieloides supresoras derivadas (MDSC) de ingenuo y de páncreas ratones portadores del tumor mediante citometría de flujo magnético y automatizado de células activadas clasificación (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

preparation-myeloid-derived-suppressor-cells-mdsc-from-naive

Research

JoVE Journal

Medicine

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

26.1K vistas.  University of South Florida Morsani College of Medicine. Este es un método rápido y completo de inmunofenotipo mieloide derivados de células supresoras (MDSC) y enriquecedora Gr-1 + Leucocitos de bazos de ratones. Este método utiliza la citometría de flujo y Celular AutoMACS clasificación para enriquecer viables Gr-1 + Leucocitos antes de FACS clasificación de MDSC para su uso En vivo Y In vitro Ensayos.

Video: Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Procesamiento de tejido primario Tumor cerebral para ensayos con células madre y flujo de Ordenación

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Investigación

Medicina

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Diferenciación de aislamiento, caracterización y comparativo de las células madre derivadas de la Pulpa Dental de los dientes permanentes mediante dos métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Paciente celulares derivadas Cultura y aislamiento de CD133<sup&gt; +</sup&gt; Putativos células madre del cáncer de melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Ensayo de alto rendimiento basado en células citometría de flujo para detectar anticuerpos a la N-metil-D-aspartato receptor o dopamina-2 del receptor en suero humano

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Adaptación de semiautomatizado de células tumorales circulantes (CTC) Ensayos para aplicaciones clínicas y de investigación preclínica

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

Validación de nanocuerpo y basados ​​en anticuerpos<em&gt; En Vivo</em&gt; Experimentos tumor de xenoinjerto NIRF-de formación de imágenes en ratones utilizando<em&gt; Ex Vivo</em&gt; Citometría de flujo y microscopía

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Aislamiento de células endoteliales linfático humano por Multi-parámetro de clasificación de células activadas por fluorescencia

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

El estudio de cáncer de páncreas Tallo Características de las células para el desarrollo de nuevas estrategias de tratamiento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. Determining the real time state of a microbial cell goes beyond live or dead categories, as microbes can exist in a dormant state, whereby cell division and metabolic activities are reduced. Given the need for detection and quantification of microbes, flow cytometry (FCM) with molecular probes provides a rapid and accurate method to help determine overall population viability. By using SYTOX Green and SYTOX Orange in the model cyanobacteria Microcystis aeruginosa to detect membrane integrity, we develop a transferable method for rapid indication of single cell mortality. The molecular probes used within this journal will be referred to as green or orange nucleic acid probes respectively (although there are other products with similar excitation and emission wavelengths that have a comparable modes of action, we specifically refer to the fore mentioned probes). Protocols using molecular probes vary between species, differing principally in concentration and incubation times. Following this protocol set out on M.aeruginosa the green nucleic acid probe was optimized at concentrations of 0.5 &#181;M after 30 min of incubation and the orange nucleic acid probe at 1 &#181;M after 10 min. In both probes concentrations less than the stated optimal led to an under reporting of cells with membrane damage. Conversely, 5 &#181;M concentrations and higher in both probes exhibited a type of non-specific staining, whereby &#39;live&#39; cells produced a target fluorescence, leading to an over representation of &#39;non-viable&#39; cell numbers. The positive controls (heat-killed) provided testable dead biomass, although the appropriateness of control generation remains subject to debate. By demonstrating a logical sequence of steps for optimizing the green and orange nucleic acid probes we demonstrate how to create a protocol that can be used to analyse cyanobacterial physiological state effectively.

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry

Microbial populations contain substantial cell heterogeneity, which can dictate overall behavior. Molecular probe analysis through flow cytometry can determine physiological states of cells, however its application varies between species. This study provides a protocol to accurately determine cell mortality within a cyanobacterium population, without underestimating or recording false positive results.

Molecular Sonda Optimización para determinar la mortalidad celular en una fotosintética Organismo (<em&gt; Microcystis aeruginosa</em&gt;) Usando citometría de flujo

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.