Preparar os Reagentes <ol><li> Faça um buffer contendo PBS com BSA (0,5%), e 2 mM EDTA. Porque as bolhas de ar podem bloquear colunas de separação MACS, o buffer deve ser desgaseificado e armazenado a 2-8 ° C antes do uso. </li><li> Usamos meio RPMI 1640 contendo soro do mouse 5%. O meio de cultura não deve conter BSA ou FCS, uma vez que estes compostos irão alterar a especificidade de estimulação celular. </li><li> A IL-17 Rato Ensaio Secreção - Enriquecimento Celular e Kit de Detecção da Miltenyi Biotec-. O kit contém os seguintes componentes: a IL-17 Reagente Catch, a IL-17 anticorpo de detecção (Biotina), o Anti-Biotina-PE, e as microesferas de Anti-PE. </li></ol> Estimulando a esplenócitos <ol><li> Este protocolo é realizado na presença de um controle negativo e positivo, como esplenócitos unstimulated e um contracoloração para células T. Este protocolo é realizado utilizando técnica estéril. </li><li> Preparar uma suspensão única célula de esplenócitos de camundongo que foram isolados usando o gentleMACS Dissociator ™. A concentração de células deve ser determinada através de contagem das células. Agregar as células em 200 × g por 10 minutos em temperatura ambiente. </li><li> Após centrifugação, aspirar o sobrenadante fora do pellet utilizando uma pipeta. Não decantar o tubo para evitar a perda de sedimento. </li><li> Agora, voltar a suspender as células em meio de cultura e, em seguida, adicionar a um poço. Adicionar média suficiente para uma concentração de 10 milhões de células por mL e cinco milhões de células por centímetro quadrado. </li><li> Para estimular uma resposta imune em nossas células ressuspenso, acrescentamos ionomicina (1 mg / mL) e PMA (10 ng / mL) para a amostra e misture delicadamente a solução pipetando para cima e para baixo. Em seguida, os poços são rotulados em conformidade. </li><li> Agora, vamos incubar nossas células durante 3 horas a 37 ° C sem mistura para iniciar o período de estimulação. Proceder à análise de IL-17 três horas desde o início da estimulação, de modo plano nesse sentido. </li><li> Para interromper a secreção, as células são colocadas em gelo e coletamos as células estimuladas por delicadamente pipetando para cima e para baixo com tampão de frio. Células são então transferidos do bem a um tubo e são lavados uma segunda vez. </li><li> Para garantir que todas as células são coletadas, é uma boa idéia para verificar o seu prato sob um microscópio. Se as células ainda permanecem conectados, você pode coletar as células restantes lavando o prato com tampão de frio. Qualquer aglomerados de células na sua suspensão de células pode ser removido usando os filtros de pré-separação. </li></ol> Rotulagem as células com reagente Trave <ol><li> É fundamental notar, que este teste funciona perfeitamente se inferior a 2% de IL-17 são células secretoras de presente. </li><li> Se a concentração de IL-17 células secretoras deverá ser superior a 2% ajustar volumes em conformidade. <img src="/files/ftp_upload/old/1037_2008_12_8_6/figure8.png" alt="figura 8" width="500" height="162" /></li><li> Uma armadilha potencial deste procedimento é a contaminação cruzada do Reagente Catch, que pode ocorrer durante a rotulagem quando o anticorpo bi-específico liga-se a uma célula T não secretores e armadilhas IL-17 secretada a partir de um linfócito vizinhos, gerando falsos positivos. </li><li> A fim de contornar este problema, é fundamental para as células esfriar antes de rotulagem e trabalhar com buffer de frio para lenta difusão de IL-17 e evitar essa contaminação cruzada. Além de manter as células frio, eles devem ser mantidos a uma concentração definida. </li><li> Para iniciar o procedimento de rotulagem, usamos 10 milhões de células em um tubo de 15 ml que pode ser fechado. Se os números mais altos de células têm de ser utilizados, simplesmente escala de todos os volumes em conformidade. Uma vez que a concentração celular ideal foi obtida, lave as células, adicionando 10 ml de tampão frio. </li><li> Girar as células em 300 × g por 10 minutos em centrífuga refrigerada (2-8 ° C). </li><li> Após centrifugação, aspirar o sobrenadante completamente usando uma pipeta. Não decantar o sobrenadante, pois isso levará a perda de células e volumes imprecisa. Repita o passo de lavagem, que consiste na adição de 10ml de solução de frio, a centrifugação, e aspiração. </li><li> Agora que temos uma pelota de pureza desejado, ressuspender as células em 80 mL de meio de cultura frio. Para classificá-los, vamos agora adicionar 20 mL de Reagente rato Pegue IL-17. </li><li> Incubar as células por 5 minutos no gelo. </li><li> Após o período de incubação 5 minutos no gelo, retire o tubo e diluir as células em 10 ml de 37 C ° média morna. Em seguida, prenda o tubo na Rotator Tubo MACSmix e incubar os tubos a 37 ° C por 45 min em movimento contínuo. Aumentando a temperatura para 37 ° C vai re-iniciar a secreção de citocinas. </li></ol> Rotulagem as células com IL-17 anticorpo de detecção (Biotina) e Anti-Biotina-PE <ol><li> Após o período de secreção de 45 minutos a 37 ° C, colocar o tubo imediatamente em gelo. Isto irá parar a secreção de citocinas. A partir daqui é fundamental para manter as células no gelo.Trabalhando com tampão frio vai evitar a contaminação cruzada do Reagente Catch. </li><li> Encha o tubo com tampão de frio e centrifugar a 300xg a 2-8 ° C por 10 min. Aspirar o sobrenadante completamente. Repita o passo lavar mais uma vez. </li><li> O pellet celular é ressuspenso em 80 mL de tampão frio eo tubo é colocado sobre o gelo. </li><li> Para evitar a ligação inespecífica do anticorpo, recomendamos a adição de 10μl Reagente FcR Bloqueio e incubar no gelo por 5 min. Então adicionar 20 mL de anticorpo de detecção do rato IL-17 (Biotina), que é conjugado a biotina e incubar por 10 minutos no gelo. </li><li> Mais uma vez, lave as células como fizemos antes, adicionando 10 mL de tampão frio e centrifugar a 300xg a 2-8 ° C por 10 min. </li><li> Agora aspirar o sobrenadante e ressuspender o pellet celular em 80 mL de tampão frio. </li><li> Adicionar 20 mL de nosso anticorpo secundário, Anti-Biotina-PE. </li><li> Misture as células e solução de anticorpo secundário, e novamente incubar os tubos durante 10 minutos no gelo. </li><li> Uma vez que esta segunda incubação é completa, lavar as células com 10 mL de tampão fria e centrifugação. </li><li> O pellet pode agora ser ressuspendida em 500 mL de tampão frio. </li><li> Se as células têm que ser separados para análise a jusante, podem ser rotulados com magneticamente Anti-PE-micro e separada manualmente ou automatizado usando MACS Colunas e Separadores de MACS. </li><li> Completamos agora a rotulagem dos esplenócitos e estão prontos para análise. </li></ol> Análise FACS <ol><li> Para nossa análise, vamos comparar os esplenócitos estimulados e desestimulados por citometria de fluxo. </li><li> Antes da análise de citometria de fluxo, as células são coradas com iodeto de propídio a 0,5 mcg / mL para deixar de fora as células mortas. O Analyzer ™ MACSQuant estava carregado com 200.000 células para cada amostra e agora estamos indo olhar os gráficos de dispersão de reatividade de anticorpos relativa nas amostras. Duas considerações importantes para a análise de células raras - como a IL-17 células positivas - por citometria de fluxo são: <ul><li> definição de um portão em linfócitos na dispersão frontal versus gráfico de dispersão lado </li><li> gating e as células mortas (coradas com iodeto de propídio) e células-B (que pode causar coloração de fundo inespecífica) para aumentar ainda mais a sensibilidade de detecção </li></ul></li><li> Usamos CD4-APC anticorpos para detectar células T e os anticorpos CD45R/B220-PerCP de detectar as células B. Nós vemos as propriedades de dispersão para frente e laterais das amostras e aplicar um portão sobre a população de linfócitos. </li><li> Um segundo portão foi aplicada a ver as células mortas e as células manchadas manchado B (eixo Y). Estas células são excluídos da análise de células T. </li><li> Neste exemplo, que é essencialmente nosso controle negativo, podemos ver que há muito poucos IL-17 CD4 T secretoras de células positivas em amostras não estimuladas - cerca de 0,003% (Figura 4). Olhando para a população de células T estimuladas, podemos ver um grande número de células CD4 positivas T que secretam IL-17 - cerca de 0,367% (Figura 5A) antes da separação e 60,76% após a separação magnética com MACS Tecnologia (Figura 5B). </li></ol>

Todos os protocolos e fichas de dados estão disponíveis em www.miltenyibiotec.com. Advertências Os reagentes contêm azida de sódio. Sob condições ácidas azida de sódio produz ácido hidrazóico, que é extremamente tóxico. Os compostos de azida deve ser diluído com água corrente antes de descartar. Estas precauções são recomendadas para evitar depósitos nas canalizações onde se podem desenvolver condições explosivas.

Células TH17 desempenhar um papel integral na imunidade adaptativa e da resposta inflamatória e são um componente fundamental na condução inflamação auto-imune. Este vídeo documenta como usar mouse Miltenyi de IL-17 Assay Secreção - Enriquecimento Celular e Kit de Detecção (PE). Ao fazer este procedimento é importante ter uma estimativa da freqüência de IL-17 em células secretoras de sua preparação célula inicial. A concentração de célula apropriada durante o período de rotulagem e secreção de citoquinas evita a contaminação cruzada por outras células em sua suspensão e garante resultados confiáveis.

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		<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>
		</div>

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

O MACS citocinas tecnologia Assay Secreção permite a detecção de citocinas secretadas no nível única célula e isolamento sensível viável células secretoras de citocinas. Para rotular IL-17 células secretoras de uma suspensão única célula de esplenócitos mouse está preparado e estimulado a 37 ° C com PMA / ionomicina para induzir a secreção de citocinas. Para parar células secreção são então colocados em gelo e são expostos à IL-17 Reagente Catch a anticorpos bi-específicos que se liga ao CD45 na superfície celular dos leucócitos e IL-17, uma vez que é secretada e pegou perto da superfície da célula. Secreção é então re-iniciados, aumentando a temperatura para 37 ° C e IL-17 é preso pelo reagente Catch. Secreção é, então, parou de novo, colocando as células no gelo. Para detectar a IL-17 presos, as células são incubadas com IL-17 específicos segundo anticorpo conjugado a biotina e um anticorpo anti-biotina-PE. Células podem agora ser analisadas diretamente por citometria de fluxo ou preparados para isolamento e enriquecimento através da rotulagem posterior com Anti-PE microesferas 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 procedimento descreve a detecção e isolamento de leucócitos do mouse TH17 que ativamente secretam IL-17 com a estimulação.

Detecção e isolamento de células viáveis ​​rato IL-17 Secretoras de T

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 visualizações.  Miltenyi Biotec,GmbH.  ChineseVersion JapaneseVersion 

Vídeo: 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.

Isolamento de células T CD4 + a partir dos nós linfáticos rato Usando Miltenyi Purificação 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 ...

Biologia

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.

Isolamento e derivação de células embrionárias de rato 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.

Isolamento de Valvular Células Endoteliais

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.

Isolamento de Células-Tronco do rato das Glândulas Salivares

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.

Isolamento de células basais e células Duct submucosa Glândulas de Rato Traquéia

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.

Isolamento e Cultura do mouse primárias células pancreáticas acinares

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.

Isolamento de Viável multicelular Glândulas de tecido da planta carnívora, Nepenthes</i

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.

Isolamento e cultura primária de células de camundongo aórtica endoteliais

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.

Isolamento de células de camundongo Coronária endoteliais

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.

Detecção e isolamento de células viáveis ​​rato IL-17 Secretoras de T

Detecção e isolamento de células viáveis rato IL-17 Secretoras de T