La financiación de este proyecto fue proporcionado por la National Science subvenciones OCE1029607 Foundation (XM) y MCB0702125 (de MAM) y Gordon y Betty Moore Foundation (de MAM).

1. Procesamiento de las muestras de agua <ol><li> Filtro de 10 litros de agua del medio ambiente a través de un filtro de membrana micras de tamaño de poro para eliminar las partículas grandes y bacteriovores. Recoger el filtrado de agua en una garrafa. </li><li> Transferencia de 36 ml de filtrado a cada uno en tres tubos estériles Eppendorf (50 ml) contiene 4 ml solución preparada recientemente paraformaldehído (PFA, el 10%). Incubar durante 2 horas a temperatura ambiente para preservar las células. Recoger las células en 0,22 micras de tamaño de poro de filtros de membrana blanco por filtración al vacío. Lave el filtro de paso de 10 ml de buffer fosfato salino (PBS) a través del filtro por filtración al vacío. Etiqueta de los filtros como controles negativos y almacenarlos a -20 ° C. </li></ol> Pasos 1.3-1.4 son opcionales para el establecimiento de DOC-limitada condiciones. <ol start="3"><li> Añadir una mezcla de nitrógeno y fósforo inorgánico (5 mM NH 4 Cl, 5 M NaNO 3, y 1μM NaH 2 PO 4 concentración final) en la bombona. </li><li> Incubar en la oscuridad a la temperatura in situ durante 48 horas con agitación ocasional. </li></ol> 2. Microcosmos y el establecimiento de incubación <ol><li> Llene cada uno de los seis frascos de vidrio de 1 L con 800 ml de muestra de agua de la bombona de paso de 1,4 a establecer microcosmos. Añadir BrdU (10 mM, concentración final) en cada microcosmos. Mezclar bien. </li><li> Agregar un modelo DOC ml solución compuesta en tres de los microcosmos, estos servirán como enmiendas DOC. Añadir 1 ml de PBS estéril en los tres restantes microcosmos, estos servirán como controles sin adición. </li><li> Incubar todos los microcosmos en un agitador incubadora y se incuban en la oscuridad a la temperatura in situ mientras se agita a 100 rpm. </li><li> Recoger 36 ml de muestra de agua de cada microcosmos y la transferencia en tubos de 50 ml estériles Eppendorf en los puntos de tiempo de 0, 8, 16 y 24 horas. Inmediatamente añadir 4 ml de agua dulce PFA (10%) de los tubos de recolección y se incuban durante 2 horas a temperatura ambiente para preservar las células. </li><li> Filtro conservan las células a través de 0,22 micras de tamaño de poro de filtros de membrana de policarbonato. Lavar los filtros con 10 ml de PBS. Proceder de inmediato a la etapa siguiente o almacenar los filtros a -20 ° C. </li></ol> 3. En la inmunodetección situ para la incorporación de BrdU <ol><li> Descongele los filtros (DOC modificado muestras, los controles no-además de controles positivos y negativos) a temperatura ambiente. </li><li> Aplicar 1 ml de solución de lisozima [10 mg / ml de lisozima de clara de huevo en 100 mM Tris, 50 mM EDTA (pH = 8)] 4 para cubrir las células bacterianas en el filtro. Incubar a temperatura ambiente durante 30 minutos. Lave el filtro pasando 10 ml de PBS a través de ella, bajo succión. </li><li> Añadir 1 ml de solución de proteinasa K [2 mg / ml de proteinasa K en 100 mM Tris, 50 mM EDTA (pH = 8)] 4 para cubrir las células bacterianas en el filtro. Incubar a temperatura ambiente durante 30 minutos. Lave el filtro pasando 10 ml de PBS a través de ella, bajo succión. </li><li> Añadir una solución de exonucleasa ml [exonucleasa III (50 U / ml) en 5 mM de MgCl2 y 50 mM Tris-HCl] 5 para cubrir las células bacterianas en el filtro. Se incuba a 37 ° C durante 30 minutos. Lave el filtro pasando 10 ml de PBS a través de ella, bajo succión. </li><li> Montar el sello marco cámaras de incubación (Bio-Rad) según las instrucciones del fabricante. </li><li> Cortar el filtro en octavos con una cuchilla estéril sobre un alcohol limpiar la superficie seca. </li><li> Con unas pinzas, coloque una sección octava de un filtro en una cámara montada incubación marco de sellado (sello ocho marco cámaras se necesitan para cada muestra de filtro). El lado de la sección de filtro que contiene las células deben estar hacia arriba. La humedad en la parte posterior del filtro permite filtrar el palo a la diapositiva. Si el filtro se vuelve seca, aplicar una pequeña gota (2 l) del DIH 2 O en el centro de la cámara antes de colocar la sección de filtro en la diapositiva. </li></ol> Pasos 3.8-3.20 usar los reactivos del kit de Roche en la proliferación celular in situ, siguiendo los procedimientos Fluos modificado a partir de las instrucciones del fabricante. A excepción de PBS, todos los reactivos se proporcionan en el kit. <ol start="8"><li> Aplicar tampón de incubación suficiente (0,5% albúmina de suero bovino, 0,1% de Tween 20 en PBS), para cubrir uniformemente toda la sección de filtro en la cámara de incubación sin causar desbordamiento de una vez que el sello se aplica. </li><li> Coloque una cubierta de poliéster marco sobre el marco de la cámara de incubación. Presione hacia abajo para sellar herméticamente la cámara de incubación. Evite las burbujas de aire sobre el filtro. Se incuban las cámaras selladas durante 10 minutos a temperatura ambiente en la oscuridad. </li><li> Retire el sello de poliéster y abrir las cámaras de incubación. (Nota: la apertura de las cámaras a veces se tire hacia arriba el sello marco de la diapositiva En ese caso, prepare una nueva cámara para los siguientes pasos..) </li><li> Pipeta el tampón de incubación de una esquina de la cámara. Evite el contacto con el filtro. </li><li> Lave el filtro con cuidado pipeteando 100 l de PBS yfuera de la cámara de 3 veces. </li><li> Prepare anti-BrdU-Fluos solución de trabajo siguiendo los pasos recomendados por el fabricante inmediatamente antes del uso. </li><li> Pipetear 120 l anti-BrdU-Fluos solución de trabajo en el filtro, teniendo cuidado de cubrir uniformemente toda la superficie. </li><li> Cerrar de nuevo la cámara con una cubierta de poliéster nuevo. Evite las burbujas de aire en la parte superior del filtro. Incubar la cámara en la oscuridad a 37 ° C durante 3 horas. Este paso se etiqueta BrdU incorporado ADN in situ con isotiocianato de fluoresceína (FITC). </li><li> Quite la cubierta de poliéster y abrir la cámara de incubación. Pipeta la solución anti-BrdU-Fluos de trabajo. Lave el filtro 3 veces con PBS. </li><li> Transferir el filtro de la cámara de incubación a una superficie estéril. Cortar la sección de filtro en pequeños trozos con una cuchilla estéril. </li><li> Transferencia de las piezas de filtro en dos tubos de microcentrífuga ml, conteniendo cada uno 1 ml de PBS. Bien tapar el tubo y sellar con parafilm. Se incuba a 37 ° C y 200 rpm durante 10 minutos. </li><li> Asegurar los tubos en un vórtice y agitar a la velocidad máxima durante 5 minutos. Pipeta el sobrenadante a un tubo estéril de 15 ml Eppendorf. Repita los pasos de incubación y vortex durante 5 veces más. Combine el sobrenadante en el mismo tubo de 15 ml Eppendorf para cada muestra. Por lo general, el 80% de las células se pueden recuperar en la suspensión. </li><li> Guardar el sobrenadante con las células se resuspendieron a 4 ° C. Ordenar dentro de 2 días. </li></ol> 4. FACS análisis Un procedimiento de un citómetro de flujo BD FACSAria y el software correspondiente que se describe aquí. <ol><li> Optimizar el ajuste de los siguientes pasos del citómetro de flujo recomendado por el fabricante. Esto implica: ajustar el control de la amplificación para establecer los parámetros necesarios breakoff y el punto dulce, optimizar el retardo de láser y los factores de escala para la zona de presión de funda experimentar y optimizar la configuración de FSC y SSC tensión, el umbral de FSC, FSC escala de fluorescencia, fluorescencia voltajes PMT , etc </li><li> Ejecutar las muestras de control negativo en el citómetro de flujo (FCM) en base a la intensidad de fluorescencia de dispersión FITC y lateral (SSC). Aumentar el umbral de FITC hasta que no las células en los controles negativos se pueden visualizar por la pantalla de adquisición de FITC-SSC. </li><li> Además de ejecutar sin las muestras de control y examinar el patrón de distribución de 10.000 células sobre la base de FITC y la adquisición de cooperación Sur-Sur. Definir una puerta para incluir todas las células y designarlos como &quot;células de baja intensidad&quot; (LI). </li><li> Ejecutar DOC-modificado muestras y examinar el patrón de distribución de 10.000 células sobre la base de FITC y cooperación Sur-Sur. Algunas células se publicará en el preset puerta LI. Definir otra puerta para encerrar las células que tienen una mayor intensidad de fluorescencia (HIS) de Lis. Adquirir las estadísticas para ver la abundancia relativa de células cerradas. </li><li> Ordenar HI células en tubos de colección que contiene 500 l de PBS a &quot;purificar una gota&quot; de modo. Terminar la clasificación, cuando el número de su cuenta llega a 500.000. </li></ol> 5. Filtro de amplificación por PCR de los genes ARNr 16S Filtro de procedimientos de PCR se han modificado desde Kirchman et al 6. <ol><li> Filtro de células seleccionadas en un fondo blanco, 0,22 m de tamaño de poro, 25 mm de diámetro, filtro de membrana de policarbonato. Recorte el borde del filtro que no contiene células bacterianas usando una cuchilla estéril. Cortar el filtro en 8 trozos de igual tamaño. </li><li> Lugar de una sola pieza de filtro en un tubo de reacción de PCR, con las células hacia adentro del tubo. Añadir 45 l de agua grado PCR en el tubo de reacción de PCR. Sumergir el filtro en su totalidad en el agua. </li><li> Añadir 2 ROCHE ilustración PuReTaq Lea-To-Go bolas de PCR en el tubo de reacción de PCR, agitar brevemente. Añadir 2 l cada uno de los de avance y retroceso gen 16S rRNA primers (0,4 mM concentración final de cada cebador), tales como 27F y 1492R 7, en cada uno de los tubos de reacción de PCR. </li><li> (Opcional) Agregue 1μl solución de albúmina sérica bovina (BSA, la concentración final es 30μg/100 l) a la mezcla de reacción de PCR para ayudar a absorber los inhibidores de la amplificación. Si se opta por no añadir BSA, añadir 1μl de agua en su lugar. </li><li> Realizar la amplificación por PCR en un termociclador. Un toque por programa de PCR se recomienda, que tiene la temperatura de hibridación secuencial disminuye 62 a 52 ° C por 1 ° C por ciclo durante 11 ciclos, seguido de 15 ciclos con temperatura de anillamiento de 52 ° C. Todos los ciclos incluyen desnaturalización (95 ° C), hibridación (a los 62 a 52 ° C), y la extensión (a 72 ° C) los pasos de la duración de los años 50. Una inicial de 3 min de desnaturalización y final de 10 min de extensión paso también se incluye en el programa de PCR. </li><li> Confirmar la amplificación de PCR por electroforesis en un teñido con bromuro de etidio 1% en gel de agarosa. Los impuestos especiales los amplicones de PCR en el gel y la limpieza con el kit de QIAGEN QIAquick extracción de gel. </li><li> Realizar dos ampliaciones adicionales de PCR para cada muestra, cada vez que utiliza una sección de filtro. Después de PCR en gel Purificatide, la piscina amplificados de la misma muestra en conjunto. Purificada 16S ADNr amplificados ya están listos para una serie de análisis moleculares que permiten la identificación taxonómica, como la construcción de la biblioteca y la secuencia de clon. </li></ol> 6. Los resultados representativos: Los resultados representativos se describen mediante un estudio de putrescina-bacterias degradantes como un ejemplo. Tomaron muestras de agua de una zona costera de Georgia y procesadas siguiendo los procedimientos descritos anteriormente. FACS análisis reveló que además de putrescina provocados por el desarrollo de un grupo de bacterias con alta intensidad de fluorescencia FITC, lo que indica la alta tasa de incorporación de BrdU (Figura 1). Estas células fueron designados como las células de alta BrdU incorporación (HIS) y se esperaba que contienen en su mayoría putrescina-degradantes bacterias. Su faltaban en los controles además de no-, que sólo contenía células con bajos niveles de incorporación de BrdU (LI). LI se esperaba que contienen principalmente bacterioplancton que no podían utilizar la putrescina añadió. Su fueron ordenados en tubos separados y recogidos en los filtros de membrana. 16S rRNA amplificados del gen se obtuvieron de células de alta HI con filtro de PCR. <img src="/files/ftp_upload/2855/2855fig1.jpg" alt="Figura 1"/> Figura 1. Análisis de citometría de flujo sin adición de control y modelo compuesto modificado (putrescina como un ejemplo aquí) las muestras tomadas después de 24 h de incubación. Análisis de las células de distribución se basa en (1) la intensidad de fluorescencia de etiquetado FITC (eje x), que se relaciona positivamente con el nivel de incorporación de BrdU, y (2) dispersión lateral (SSC, eje y), que está positivamente relacionado con la célula tamaño. Puerta de la notación se basa en el nivel de incorporación de BrdU, (HI, de alta BrdU incorporación, LI, bajo BrdU incorporación). El porcentaje relativo de HI y las células LI se muestran en la puerta correspondiente.

<ol>
	<li>Pernthaler, A., Pernthaler, J., Schattenhofer, M., Amann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+DNA-synthesizing+bacterial+cells+in+coastal+North+Sea+plankton.">Identification of DNA-synthesizing bacterial cells in coastal North Sea plankton.</a> Appl. Environ. Microbiol. 68, 5728-5728 (2002).</li><li>Urbach, E., Vergin, K. L., Giovannoni, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunochemical+detection+and+isolation+of+DNA+from+metabolically+active+bacteria.">Immunochemical detection and isolation of DNA from metabolically active bacteria.</a> Appl. Environ. Microbiol. 65, 1207-12 (1999).</li><li>Mou, X. Z., Hodson, R. E., Moran, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterioplankton+assemblages+transforming+dissolved+organic+compounds+in+coastal+seawater.">Bacterioplankton assemblages transforming dissolved organic compounds in coastal seawater.</a> Environ. Microbiol. 9, 2025-2025 (2007).</li><li>Hodson, R. E., Dustman, W. A., Garg, R. P., Moran, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+PCR+for+visualization+of+microscale+distribution+of+specific+genes+and+gene+products+in+prokaryotic+communities.">In situ PCR for visualization of microscale distribution of specific genes and gene products in prokaryotic communities.</a> Appl. Environ. Microbiol. 61, 4074-4074 (1995).</li><li>Dinjens, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bromodeoxyuridine+(BrdU)+immunocytochemistry+by+exonuclease+III+(Exo+III)+digestion.">Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion.</a> Histochemistry. 98, 199-199 (1992).</li><li>Kirchman, D. L., Yu, L. Y., Fuchs, B. M., Amann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+bacterial+communities+in+aquatic+systems+as+revealed+by+filter+PCR.">Structure of bacterial communities in aquatic systems as revealed by filter PCR.</a> Aquat. Microb. Ecol. 26, 13-13 (2001).</li><li>Delong, E. F., Wickham, G. S., Pace, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+stains:+ribosomal+RNA-based+probes+for+the+identification+of+single+cells.">Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells.</a> Science. 243, 1360-1360 (1989).</li><li>Artursson, V., Jansson, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+bromodeoxyuridine+immunocapture+to+identify+active+bacteria+associated+with+arbuscular+mycorrhizal+hyphae.">Use of bromodeoxyuridine immunocapture to identify active bacteria associated with arbuscular mycorrhizal hyphae.</a> Appl. Environ. Microbiol. 69, 6208-6208 (2003).</li><li>Mou, X. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+carbon+processing+by+generalist+species+in+the+coastal+ocean.">Bacterial carbon processing by generalist species in the coastal ocean.</a> Nature. 451, 708-708 (2008).</li><li>Mou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-cytometric+cell+sorting+and+subsequent+molecular+analyses+for+culture-independent+identification+of+bacterioplankton+involved+in+dimethylsulfoniopropionate+transformations.">Flow-cytometric cell sorting and subsequent molecular analyses for culture-independent identification of bacterioplankton involved in dimethylsulfoniopropionate transformations.</a> Appl. Environ. Microbiol. 71, 1405-1405 (2005).</li><li>Dean, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+human+genome+amplification+using+multiple+displacement+amplification.">Comprehensive human genome amplification using multiple displacement amplification.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 5261-5261 (2002).</li></ol>

No hay conflictos de interés declarado.

Nuestras parejas enfoque BrdU incorporación, FACS y análisis de 16S ADNr para permitir que las especies a nivel de identificación de bacterioplancton que metabolizan los componentes individuales de DOC en los ambientes acuáticos. El ensayo de incorporación de BrdU etiquetas de las células bacterianas sobre la base de las actividades metabólicas, lo que permite el análisis sólo en las bacterias activas y por lo tanto no incluye a las células inactivas. En nuestro enfoque, la incorporación de BrdU se encuentra ...

<table border="1"><tbody><tr><td> Nombre </td><td> Tipo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> BrdU </td><td> Reactivo </td><td> Sigma </td><td> B5002-5G </td><td></td></tr><tr><td> Lisozima </td><td> Reactivo </td><td> Sigma </td><td> L6876-5G </td><td></td></tr><tr><td> Proteinasa K </td><td> Reactivo </td><td> Sigma </td><td> P2308-25MG </td><td></td></tr><tr><td> In Situ proliferación de células Kit, Fluos </td><td> Equipo </td><td> Roche </td><td> 11810740001 </td><td> Consumir más de Anti-BrdU-Fluos tampón de incubación y por la reacción que lo sugerido por el fabricante. </td></tr><tr><td> Sello marco de las cámaras de incubación </td><td> Material </td><td> Bio-Rad </td><td> SLF-1201 </td><td></td></tr><tr><td> Filtros de membrana de policarbonato (142 mm de diámetro, 1,0 m de tamaño de poro) </td><td> Material </td><td> Millipore </td><td> FALP14250 </td><td></td></tr><tr><td> Filtros de membrana de policarbonato (25 mm de diámetro, 0,2 m de tamaño de poro) </td><td> Material </td><td> Millipore </td><td> FGLP02500 </td><td></td></tr><tr><td> ilustración PuReTaq Ready-To-Go PCR Beads </td><td> Equipo </td><td> GE Healthcare </td><td> 27-9559-01 </td><td></td></tr><tr><td> QIAquick gel extracción kit </td><td> Equipo </td><td> QIAGEN </td><td> 28704 </td><td></td></tr><tr><td> FailSafe de PCR System </td><td> Equipo </td><td> EPICENTRO </td><td> FS99060 </td><td></td></tr></tbody></table>

bromodeoxyuridine (brdu) labeling and subsequent fluorescence activated cell sorting for culture-independent identification of dissolved organic carbon-degrading bacterioplankton

Los microbios son los principales agentes de mediación de la degradación de muchos de carbono orgánico disuelto (COD) sustratos en los ambientes acuáticos. Sin embargo, la identificación de los taxones de bacterias que transforman las piscinas específicas de DOC en la naturaleza supone un reto técnico. A continuación se describe un enfoque que las parejas bromodesoxiuridina (BrdU) incorporación, la fluorescencia de células activadas clasificación (FACS), y 16S ARNr de análisis basados ​​en genes molecular que permite que la cultura independiente de la identificación de bacterioplancton capaces de degradar un compuesto DOC específicas en los ambientes acuáticos. Microcosmos por triplicado bacterioplancton están preparados para recibir tanto BrdU y un compuesto modelo DOC (DOC modificaciones), o sólo BrdU (sin adición de control). Sustitutos de BrdU las posiciones de la timidina en el ADN de nueva síntesis bacteriana y BrdU marcado de ADN puede ser fácilmente immunodetected 1,2. A través de una de 24 horas de incubación, bacterioplancton que son capaces de utilizar el compuesto añadido DOC se espera que se activan selectivamente, y por lo tanto, tienen mayores niveles de incorporación de BrdU (células HI) que no responde a las células en las enmiendas DOC y las células no- Además de los controles (bajo recuento de incorporación de BrdU, las células LI). Después de inmunodetección de fluorescencia, las células se distinguen HI y separadas físicamente de las células LI por clasificación celular activado por fluorescencia (FACS) 3. Ordenado DOC-respuesta (células HI) se extraen de ADN y taxonómicamente identificados a través de 16S rRNA gen posterior análisis basados ​​en PCR como, la construcción de la biblioteca y la secuencia de clon.

Watch this Scientific Journal Video about Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degradin

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Bacterioplancton ambientales se incuban con un modelo del carbono orgánico disuelto (COD) y un reactivo compuesto de ADN de etiquetado, bromodesoxiuridina (BrdU). Después, DOC-degradantes células se separan de la comunidad a granel en base a su elevada incorporación de BrdU utilizando fluorescencia clasificación de células activadas (FACS). Estas células son identificadas por los posteriores análisis molecular.

Bromodeoxyuridine (BrdU) El etiquetado y posterior clasificación de células activadas fluorescencia para la Cultura independiente de la identificación de carbono orgánico disuelto degradación bacterioplancton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

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Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

20.8K vistas.  Kent State University. Bacterioplancton ambientales se incuban con un modelo del carbono orgánico disuelto (COD) y un reactivo compuesto de ADN de etiquetado, bromodesoxiuridina (BrdU). Después, DOC-degradantes células se separan de la comunidad a granel en base a su elevada incorporación de BrdU utilizando fluorescencia clasificación de células activadas (FACS). Estas células son identificadas por los posteriores análisis molecular.

Video: Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

Generación de humanos CD40 activado por células B

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Investigación

Biología

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

Fluorescencia de células activadas por la clasificación de los protoplastos de la planta

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Lípidos vesículas mediada por cromatografía de afinidad utilizando magnética clasificación de células activadas (LIMACS): un nuevo método para analizar la interacción de proteínas y lípidos

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Análisis específico de célula de<em&gt; Arabidopsis</em&gt; Hojas usando citometría de flujo

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Optimizado manchas y Proliferación métodos de modelización para la vigilancia del uso de tintes División Celular Celulares de seguimiento

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

Aislamiento de fibroblastos normales y el cáncer asociado-de los tejidos frescos por Clasificación células activadas por fluorescencia (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations.

Yeast, Saccharomyces cerevisiae, has been a key model organism to identify and study genes regulating the biogenesis and functions of the endosomal system. Here we present a detailed protocol for the specific labeling of the endosomal compartments for ultrastructural studies.

Nanogold Etiquetado del Sistema Endosomal Levadura de análisis ultraestructurales

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Etiquetado específico de secuencia de ácidos nucleicos y proteínas con metiltransferasas y el cofactor Análogos

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Célula activada por el magnético clasificación estrategias para aislar y purificar sinovial líquido mesenquimales células madre derivadas de un modelo de conejo

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

Microaspiración de células únicas como estrategia alternativa a la clasificación celular activada por fluorescencia para la separación de mezclas de virus gigantes

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...