Preparación de los reactivos <ol><li> Hacer un tampón que contiene PBS con BSA (0,5%), y 2 mM EDTA. Debido a que las burbujas de aire pueden bloquear columnas MACS separación, el buffer debe ser desgasificado y almacenado a 2-8 ° C antes de su uso. </li><li> Usamos medio RPMI 1640 que contenía 5% de suero de ratón. El medio de cultivo no debe contener BSA o FCS, ya que estos compuestos alteran la especificidad de la estimulación de las células. </li><li> La IL-17 de ratón Ensayo secreción - Enriquecimiento de células y kit de detección de Miltenyi Biotec-. El kit contiene los siguientes componentes: la IL-17 Reactivo de captura, la IL-17 de detección de anticuerpos (Biotina), el anti-biotina-PE, y el MicroBeads Anti-PE. </li></ol> Estimular los esplenocitos <ol><li> Este protocolo se realiza en presencia de un control positivo y negativo, como esplenocitos estimulados y de contraste de las células T. Este protocolo se lleva a cabo mediante una técnica estéril. </li><li> Prepare una suspensión de células individuales de esplenocitos de ratón que fueron aislados usando el disociador gentleMACS ™. La concentración de células debe ser determinado a través de recuento de células. Que sedimenten las células a 200 g durante 10 minutos a temperatura ambiente. </li><li> Después de la centrifugación, se aspira el sobrenadante de la bolita con una pipeta. No se decanta el tubo para evitar la pérdida de la pastilla. </li><li> Ahora, volver a suspender las células en el medio de cultivo y luego agregar a un pozo. Añadir medio suficiente para una concentración de diez millones de células por ml y cinco millones de células por centímetro cuadrado. </li><li> Para estimular una respuesta inmunológica en las células resuspendidas, añadimos ionomicina (1 mg / ml) y PMA (10 ng / mL) a la muestra y mezclar la solución pipeteando suavemente hacia arriba y hacia abajo. A continuación, los pozos están etiquetados en consecuencia. </li><li> Ahora, vamos a incubar las células durante 3 horas a 37 ° C sin que se mezclen para iniciar el período de estimulación. Proceder a la IL-17 análisis de 3 horas desde el inicio de la estimulación, por lo que planificar en consecuencia. </li><li> Para detener la secreción de las células se colocan sobre hielo y recoger las células estimuladas con cuidado pipeteando arriba y abajo con tampón frío. Las células se transfieren desde el pozo a un tubo y se lavan por segunda vez. </li><li> Para asegurarse de que todas las células se recogen, es una buena idea revisar su plato con un microscopio. Si las células siguen unidos, usted puede recoger las células que quedan por aclarar el plato con un tampón frío. Cualquier grupo de células en suspensión de células se pueden eliminar utilizando los filtros de pre-separación. </li></ol> Etiquetado de las células con el reactivo de captura <ol><li> Es de suma importancia señalar, que esta prueba funciona de manera óptima si menos del 2% de células IL-17-secretoras están presentes. </li><li> Si la concentración de las células secretoras de IL-17 se espera que sea superior al 2% ajustar el volumen en consecuencia. <img src="/files/ftp_upload/old/1037_2008_12_8_6/figure8.png" alt="la figura 8" width="500" height="162" /></li><li> Un riesgo potencial de este procedimiento es la contaminación cruzada de los reactivos de captura, lo que puede ocurrir durante el etiquetado cuando el anticuerpo bi-específicos se une a una célula T no secretor y las trampas de la IL-17 secretada por un linfocito vecinos, generando así los falsos positivos. </li><li> Con el fin de evitar este problema, es fundamental que las células se enfríe antes de que el etiquetado y el trabajo con tampón frío para disminuir la difusión de la IL-17 y evitar esta contaminación cruzada. Además de mantener las células en frío, que deben mantenerse a una concentración definida. </li><li> Para comenzar el procedimiento de etiquetado, utilizamos diez millones de células en un tubo de 15 ml con cierre. Si es mayor el número de células tienen que ser utilizados, simplemente escala de todos los volúmenes en consecuencia. Una vez que la concentración óptima de la célula se ha obtenido, se lavan las células mediante la adición de 10 ml de solución tampón frío. </li><li> Centrifugar las células a 300 g durante 10 minutos en una centrífuga refrigerada (2-8 ° C). </li><li> Tras la centrifugación, aspirar el sobrenadante por completo con una pipeta. No se decanta el sobrenadante, ya que dará lugar a la pérdida de células y de los volúmenes imprecisos. Repita el paso de lavado, que consiste en agregar 10 ml de tampón frío, centrifugación y aspiración. </li><li> Ahora que tenemos una bolita de la pureza deseada, resuspender las células en 80 L de medio de cultivo frío. Para etiquetar ellos, ahora vamos a añadir 20 l de reactivo de captura del ratón IL-17. </li><li> Se incuban las células durante 5 minutos en hielo. </li><li> Después del período de incubación de 5 minutos en hielo, retire el tubo y diluir las células en 10 ml de 37 º C a medio caliente. A continuación, fijar el tubo en el rotador del tubo MACSmix e incubar el tubo a 37 ° C durante 45 minutos en movimiento continuo. El aumento de la temperatura a 37 ° C se reiniciará la secreción de citoquinas. </li></ol> Etiquetado de las células con anticuerpos de detección de IL-17 (biotina) y anti-biotina-PE <ol><li> Después del período de la secreción de 45 minutos a 37 ° C, se coloca el tubo en hielo inmediatamente. Esto evitará que la secreción de citoquinas. A partir de aquí es muy importante para mantener las células en hielo.Trabajar con un tampón frío se evitará la contaminación cruzada de los reactivos de captura. </li><li> Llenar el tubo con tampón frío y centrifugar a 300xg a 2-8 º C durante 10 min. Aspirar el sobrenadante por completo. Repita el paso de lavado una vez más. </li><li> El pellet de células se resuspende en 80 l de tampón frío y el tubo se coloca en el hielo. </li><li> Para evitar la unión inespecífica del anticuerpo, se recomienda la adición de reactivos FcR 10μl de bloqueo e incubar en hielo durante 5 min. Luego se añaden 20 L de ratón de detección de anticuerpos IL-17 (biotina), que se conjuga con biotina y se incuba durante 10 minutos en hielo. </li><li> Una vez más, lavar las células como lo hemos hecho antes mediante la adición de 10 ml de tampón frío y centrifugar a 300xg a 2-8 º C durante 10 min. </li><li> Ahora aspirar el sobrenadante y resuspender el botón celular en 80 l de tampón frío. </li><li> Añadir 20 l de nuestra secundaria de anticuerpos, anti-biotina-PE. </li><li> Mezclar las células y la solución de anticuerpo secundario, y de nuevo se incuba el tubo durante 10 minutos en hielo. </li><li> Una vez que esta segunda incubación se completa, se lavan las células con 10 ml de tampón frío y centrifugar. </li><li> El pellet de ahora puede ser resuspendido en 500 L de tampón frío. </li><li> Si las células tienen que ser separados para el análisis de aguas abajo, pueden ser marcadas magnéticamente con Anti-PE-MicroBeads y separados de forma manual o automática utilizando columnas MACS y separadores de MACS. </li><li> Hemos completado el etiquetado de los esplenocitos y están listos para su análisis. </li></ol> FACS análisis <ol><li> Para nuestro análisis, vamos a comparar los esplenocitos estimulados y no estimulados por citometría de flujo. </li><li> Antes del análisis de citometría de flujo, las células se tiñeron con yoduro de propidio a 0,5 mg / ml a la puerta de las células muertas. El MACSQuant Analyzer ™ se ha cargado con 200.000 células por cada muestra y que ahora se va a mirar los gráficos de dispersión de la reactividad de los anticuerpos en relación a las muestras. Dos consideraciones importantes para el análisis de células raras - como la IL-17 células positivas - por citometría de flujo son los siguientes: <ul><li> establecimiento de una puerta en los linfocitos de la dispersión frontal versus gráfico de dispersión lateral </li><li> gating y las células muertas (teñidas con yoduro de propidio) y las células B (que puede causar la tinción de fondo inespecífica) para mejorar aún más la sensibilidad de detección </li></ul></li><li> Hemos utilizado CD4-APC anticuerpos para detectar las células T y los anticuerpos CD45R/B220-PerCP para detectar las células B. Vemos las propiedades de dispersión frontal y lateral de las muestras y aplicar una puerta en la población de linfocitos. </li><li> Una segunda puerta se aplicó para ver las células teñidas muertos y células teñidas B (eje Y). Estas células son excluidos del análisis de células T. </li><li> En este ejemplo, que es esencialmente nuestro control negativo, podemos ver que hay células IL-17 CD4 secretan muy pocos positivos en las muestras de T estimuladas - aproximadamente 0,003% (Figura 4). En cuanto a la población de células T estimuladas, podemos ver un gran número de células CD4 positivas las células T que secretan IL-17 - de 0.367% (Figura 5) antes de la separación y el 60,76% después de la separación magnética con tecnología MACS (Figura 5). </li></ol>

Todos los protocolos y hojas de datos están disponibles en www.miltenyibiotec.com. Advertencias Los reactivos contienen azida de sodio. En condiciones ácidas azida de sodio en ácido hidrazoico, que es extremadamente tóxico. Los compuestos de azida deben diluirse con agua antes de desecharlos. Se recomiendan estas precauciones para evitar depósitos en las tuberías, en las que pueden explotar.

Las células TH17 desempeñan un papel integral en la inmunidad adaptativa y la respuesta inflamatoria y son un componente clave en la conducción de la inflamación autoinmune. Este video documenta el uso del ratón Miltenyi de IL-17 Ensayo de secreción - Enriquecimiento de células y kit de detección (PE). Cuando se realiza este procedimiento, es importante tener una estimación de la frecuencia de células IL-17-secreción en su preparación de células de partida. La concentración de células apropiadas durante el período de etiquetado y la secreción de citoquinas evita la contaminación cruzada por otras células en la suspensión y asegura resultados fiables.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Mouse IL-17 Secretion Assay Cell Enrichment and Detection Kit (PE) </td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-094-213</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_98_1005_Mouse_IL_17_Secretion_Assay_Cell_Enrichment_and_Detection_Kit.aspx">http://www.miltenyibiotec.com/en/PG_98_1005_Mouse_IL_17_Secretion_Assay_Cell_Enrichment_and_Detection_Kit.aspx</a>
</td>
</tr>
<tr>
<td>RPMI 1640</td>
<td>Medium</td>
<td>Miltenyi Biotec</td>
<td>130-091-440</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_243_293_RPMI_1640.aspx">http://www.miltenyibiotec.com/en/PG_243_293_RPMI_1640.aspx</a></td>
</tr>
<tr>
<td>CD4-APC</td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-091-611</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_599_327_CD4.aspx">http://www.miltenyibiotec.com/en/PG_599_327_CD4.aspx</a></td>
</tr>
<tr>
<td>CD8a-FITC</td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-091-605</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_599_328_CD8a.aspx">http://www.miltenyibiotec.com/en/PG_599_328_CD8a.aspx</a></td>
</tr>
<tr>
<td>Propidium Iodide Solution</td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-093-233</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx">http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx</a></td>
</tr>
<tr>
<td>FcR Blocking reagent, mouse </td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-092-575</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_601_435_FcR_Blocking_Reagent_mouse_.aspx">http://www.miltenyibiotec.com/en/PG_601_435_FcR_Blocking_Reagent_mouse_.aspx</a></td>
</tr>
<tr>
<td>Dead Cell Removal Kit </td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-090-101</td>
<td>&nbsp;</td>
<tr>
<td>MACSmix Tube Rotator</td>
<td>Instrument</td>
<td>Miltenyi Biotec</td>
<td>130-090-753</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx">http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx</a></td>
</tr>
<tr>
<td>gentleMACS&#x2122; Dissociator</td>
<td>Instrument</td>
<td>Miltenyi Biotec</td>
<td>130-093-235</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx">http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx</a></td>
</tr>
<tr>
<td>autoMACS Pro Starting Kit</td>
<td>Instrument</td>
<td>Miltenyi Biotec </td>
<td>130-092-545</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_651_679_autoMACS_Pro_Starting_Kit.aspx">http://www.miltenyibiotec.com/en/PG_651_679_autoMACS_Pro_Starting_Kit.aspx</a></td>
</tr>
<tr>
<td>MACS Columns</td>
<td>Tool</td>
<td>Miltenyi Biotec</td>
<td>&nbsp;</td>
<td><a href="http://www.miltenyibiotec.com/en/NN_115_Columns_at_a_glance.aspx">http://www.miltenyibiotec.com/en/NN_115_Columns_at_a_glance.aspx</a></td>
</tr>
<tr>
<td>MACS Separator</td>
<td>Tool</td>
<td>Miltenyi Biotec</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
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detection and isolation of viable mouse il-17-secreting t cells

La secreción de citocinas MACS tecnología de ensayo permite la detección de citocinas en el nivel de células individuales y el aislamiento sensible de la secreción de citoquinas viable de células. Con el fin de la etiqueta IL-17-secretora de las células, una suspensión de células individuales de esplenocitos de ratón se prepara y se estimuló a 37 ° C con PMA / ionomicina para inducir la secreción de citoquinas. Para detener la secreción de las células se colocan en hielo y se exponen a la IL-17 reactivo captura un anticuerpo bi-específicos que se une a CD45 en la superficie celular de leucocitos y de IL-17, ya que es secretada y capturado cerca de la superficie celular. La secreción se vuelve a iniciadas por el aumento de la temperatura a 37 ° C y IL-17 es atrapado por el reactivo de captura. La secreción de entonces se detuvo de nuevo, mediante la colocación de las células en hielo. Para detectar la IL-17 atrapadas, las células se incubaron con un IL-17-específicas segundo anticuerpo conjugado con biotina y un anticuerpo anti-biotina-PE. Las células pueden ahora ser analizado directamente por citometría de flujo o preparados para el aislamiento y enriquecimiento de su posterior marcado con anti-PE MicroBeads conjugados.

Watch this Scientific Journal Video about Detection and Isolation of Viable Mouse IL-17-Secreting T Cells at JoVE.com

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

Este procedimiento describe la detección y aislamiento de los leucocitos de ratón TH17 que secretan activamente IL-17 a la estimulación.

Detección y aislamiento de células viables Ratón T IL-17 Secretoras

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable ...

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detection-and-isolation-of-viable-mouse-il-17-secreting-t-cells

Research

JoVE Journal

Biology

11.1K vistas.  Miltenyi Biotec,GmbH.  ChineseVersion JapaneseVersion 

Video: Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Aislamiento de células T CD4 + de los ganglios linfáticos del ratón utilizando Miltenyi Purificación MACS

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Investigación

Biología

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

El aislamiento y la derivación de células embrionarias de ratón Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Aislamiento de células endoteliales valvular

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Aislamiento de células de la glándula salival del ratón madre

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

Aislamiento de células basales y de células submucosas conducto de la glándula de ratón tráquea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

Aislamiento y cultivo de ratón primarias células acinares pancreáticas

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.
Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.
In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Plants glands are specialized structures responsible for the biosynthesis and secretion of many compounds involved in interactions with the biotic environment. To enable studies on their molecular and biochemical features, a mechanical micropreparation technique was established in order to isolate single metabolically active glands, here from the carnivorous plant Nepenthes.

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Aislamiento y cultivo primario de células endoteliales aórticas de ratón

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Aislamiento de células endoteliales de ratón coronaria

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.