Preparazione dei reagenti <ol><li> Fai un buffer contenente PBS con BSA (0,5%), e 2 mM EDTA. Perché le bolle d&#39;aria in grado di bloccare colonne di separazione MACS, il buffer deve essere degassata e conservato a 2-8 ° C prima dell&#39;uso. </li><li> Noi usiamo RPMI 1640 medium contenente siero del mouse 5%. Il terreno di coltura non deve contenere BSA o FCS, in quanto questi composti alterano la specificità della stimolazione delle cellule. </li><li> L&#39;IL-17 mouse Assay secrezione - Arricchimento delle cellule e Detection Kit-da Miltenyi Biotec. Il kit contiene i seguenti componenti: l&#39;IL-17 reagente di cattura, la IL-17 Individuazione di anticorpi (biotina), l&#39;anti-biotina-PE, e l&#39;Anti-PE microsfere. </li></ol> Stimolare il splenociti <ol><li> Questo protocollo è eseguita in presenza di un controllo negativo e positivo, come splenociti non stimolate e di contrasto per le cellule T. Questo protocollo è eseguita con tecnica sterile. </li><li> Preparare una sospensione singola cella di splenociti mouse che sono stati isolati utilizzando il dissociatore gentleMACS ™. La concentrazione di cellule devono essere predeterminati attraverso il conteggio delle cellule. Agglomerare le cellule a 200 × g per 10 minuti a temperatura ambiente. </li><li> Dopo la centrifugazione, aspirare il supernatante fuori il pellet con una pipetta. Non decantare il tubo per evitare la perdita del pellet. </li><li> Ora, risospendere le cellule nel mezzo di coltura e quindi aggiungere ad un pozzo. Aggiungere mezzo sufficiente per una concentrazione di dieci milioni di cellule per mL e cinque milioni di cellule per centimetro quadrato. </li><li> Per stimolare una risposta immunitaria nelle nostre cellule risospese, aggiungiamo ionomicina (1 mg / mL) e PMA (10 ng / ml) al campione e mescolare la soluzione pipettando gentilmente su e giù. Poi i pozzi sono debitamente etichettati. </li><li> Ora, noi incubare le nostre cellule per 3 ore a 37 ° C senza miscelazione per iniziare il periodo di stimolazione. Procedere alla IL-17 analisi 3 ore dalla comparsa dei stimolazione, in modo da pianificare di conseguenza. </li><li> Per interrompere la secrezione, le cellule sono immessi sul ghiaccio e raccogliamo le cellule stimolate pipettando gentilmente su e giù con il tampone a freddo. Le cellule sono poi trasferiti dal bene a un tubo e vengono lavati una seconda volta. </li><li> Per garantire che tutte le cellule vengono raccolte, è una buona idea controllare il vostro piatto al microscopio. Se le cellule sono ancora attaccato, è possibile raccogliere le cellule rimanenti risciacquando il piatto con il tampone a freddo. Eventuali grumi di cellule in sospensione cellulare può essere rimosso utilizzando il pre-separazione filtri. </li></ol> Etichettare le cellule con reagente Cattura <ol><li> E &#39;fondamentale notare che questo test funziona in modo ottimale se meno del 2% di IL-17-secernenti cellule sono presenti. </li><li> Se la concentrazione di IL-17 le cellule secernenti dovrebbe essere superiore al 2% regolare i volumi di conseguenza. <img src="/files/ftp_upload/old/1037_2008_12_8_6/figure8.png" alt="figura 8" width="500" height="162" /></li><li> Un potenziale problema di questa procedura è la contaminazione incrociata dei reagenti Catch, che può verificarsi durante l&#39;etichettatura quando il bi-specifico anticorpo si lega ad un non-secernenti delle cellule T e le trappole IL-17 secreta da un linfocita vicini, generando falsi positivi. </li><li> Per aggirare questo problema, è fondamentale per le cellule raffreddare prima dell&#39;etichettatura e lavorare con tampone a freddo per rallentare la diffusione di IL-17 e di evitare la contaminazione incrociata. Oltre a mantenere le celle frigorifere, devono essere mantenuti ad una concentrazione definita. </li><li> Per iniziare la procedura di etichettatura, si usa 10.000.000 cellule in una provetta da 15 ml richiudibili. Se il numero di cellule superiore devono essere utilizzati, semplicemente scala di tutti i volumi di conseguenza. Una volta che la concentrazione cellulare ottimale è stata ottenuta, lavare le cellule con l&#39;aggiunta di 10 ml di tampone a freddo. </li><li> Spin giù le cellule a 300 × g per 10 minuti in una centrifuga refrigerata (2-8 ° C). </li><li> Dopo la centrifugazione, aspirare il surnatante completamente con una pipetta. Non decantare il sopranatante come questo porterà alla perdita di cellule e volumi impreciso. Ripetere la fase di lavaggio, che consiste di aggiungere 10 ml di tampone a freddo, centrifugazione, e aspirazione. </li><li> Ora che abbiamo un pellet di purezza desiderato, risospendere le cellule in 80 ml di terreno di coltura a freddo. Etichettarli, noi ora aggiungere 20 ml di mouse IL-17 reagente Catch. </li><li> Incubare le cellule per 5 minuti in ghiaccio. </li><li> Dopo il periodo di incubazione di 5 minuti sul ghiaccio, rimuovere il tubo e diluire le cellule in 10 ml di 37 ° C di media caldi. Poi fissare il tubo sul Tubo Rotator MACSmix e incubare il tubo a 37 ° C per 45 minuti sotto continuo movimento. L&#39;aumento della temperatura a 37 ° C ripartirà secrezione di citochine. </li></ol> Etichettare le cellule con IL-17 Individuazione di anticorpi (biotina) e anti-biotina-PE <ol><li> Dopo il periodo di secrezione 45 minuti a 37 ° C, mettere il tubo immediatamente in ghiaccio. Questo fermerà la secrezione di citochine. Da qui in poi è fondamentale per mantenere le cellule in ghiaccio.Lavorare con tampone freddo evitare la contaminazione crociata dei reagenti Catch. </li><li> Riempire il tubo con tampone a freddo e centrifugare a 300xg a 2-8 ° C per 10 min. Aspirare il surnatante completamente. Ripetere la fase di lavaggio ancora una volta. </li><li> Il pellet cellulare è risospeso in 80 ml di tampone a freddo e il tubo è posto su ghiaccio. </li><li> Per evitare il legame non specifico degli anticorpi, si consiglia l&#39;aggiunta di reagente FCR 10μl di blocco e incubare in ghiaccio per 5 min. Poi si aggiungono 20 l di mouse IL-17 Individuazione di anticorpi (biotina) che è coniugato con biotina e incubare per 10 minuti in ghiaccio. </li><li> Ancora una volta, lavare le cellule come abbiamo fatto prima con l&#39;aggiunta di 10 mL di tampone a freddo e centrifugare a 300xg a 2-8 ° C per 10 min. </li><li> Ora aspirare il supernatante e risospendere il pellet cellulare in 80 ml di tampone a freddo. </li><li> Aggiungere 20 ml di nostro anticorpo secondario, anti-biotina-PE. </li><li> Mescolare le cellule e la soluzione di anticorpo secondario, incubare e di nuovo il tubo per 10 minuti in ghiaccio. </li><li> Una volta che questa seconda incubazione è completa, lavare le cellule con 10 mL di tampone fredda e centrifuga. </li><li> Il pellet può essere risospeso in 500 ml di tampone a freddo. </li><li> Se le cellule devono essere separati per ulteriori analisi a valle, possono essere magneticamente etichettati con Anti-PE-microsfere e separati manualmente o automatizzati con colonne MACS e separatori MACS. </li><li> Ora abbiamo completato l&#39;etichettatura del splenociti e sono pronti per l&#39;analisi. </li></ol> FACS analisi <ol><li> Per la nostra analisi, metteremo a confronto i splenociti stimolati e non stimolate mediante citometria a flusso. </li><li> Prima di citometria a flusso, le cellule sono colorate con ioduro di propidio a 0,5 mg / ml per escludere le cellule morte. L&#39;analizzatore MACSQuant ™ è stato caricato con 200.000 cellule per ogni campione e che ora stanno andando a guardare le trame dispersione di reattività anticorpale relativa nei campioni. Due considerazioni importanti per l&#39;analisi di cellule rare - come IL-17 cellule positive - mediante citometria di flusso sono: <ul><li> impostazione di un cancello sulla linfociti nel scatter in avanti rispetto a scatter plot lato </li><li> gating e fuori le cellule morte (colorate con ioduro di propidio) e B-cellule (che possono provocare colorazione aspecifica di fondo) per migliorare ulteriormente la sensibilità di rilevazione </li></ul></li><li> Abbiamo usato CD4-APC anticorpi per rilevare le cellule T e l&#39;anticorpo CD45R/B220-PerCP per rilevare le cellule B. Vediamo le proprietà scatter in avanti e laterale dei campioni e applicare un gate sulla popolazione linfocitaria. </li><li> Una seconda porta è stata applicata per vedere le cellule colorate le cellule morte e B colorate (asse Y). Queste cellule sono esclusi dall&#39;analisi delle cellule T. </li><li> In questo esempio, che è essenzialmente negativo del nostro controllo, possiamo vedere che ci sono pochissimi IL-17 CD4 cellule che secernono positivo T nei campioni non stimolata - circa il 0,003% (Figura 4). Guardando alla popolazione di cellule T stimolate, possiamo vedere un numero consistente di cellule T CD4 positive che secernono IL-17 - circa 0,367% (Figura 5A) prima della separazione e 60,76% dopo la separazione magnetica con MACS Technology (Figura 5B). </li></ol>

Tutti i protocolli e le schede tecniche sono disponibili presso www.miltenyibiotec.com. Avvertenze I reagenti contengono sodio azide. In condizioni di acidità sodio azide produce acido idrazoico, che è estremamente tossico. I composti devono essere diluiti con acqua corrente prima di smaltimento. Queste precauzioni sono raccomandate per evitare depositi nelle tubazioni che esplosivo può sviluppare.

Cellule TH17 giocano un ruolo fondamentale nel immunità adattativa e la risposta infiammatoria e sono una componente chiave nel guidare l&#39;infiammazione autoimmune. Questo video documenta come utilizzare il mouse Miltenyi di IL-17 Assay secrezione - Arricchimento cellulare e kit di rilevamento (PE). Nel fare questa procedura è importante avere una stima della frequenza di IL-17-cellule che secernono nella vostra preparazione delle cellule di partenza. La concentrazione cella appropriata durante l&#39;etichettatura e la secrezione di citochine periodo impedisce la contaminazione incrociata da altre cellule in sospensione e assicura risultati affidabili.

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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</tr>
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<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>
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<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>
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<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>
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<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>
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<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>
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<td>Dead Cell Removal Kit </td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-090-101</td>
<td>&nbsp;</td>
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<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>
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<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>
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<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>
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<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>
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<td>MACS Separator</td>
<td>Tool</td>
<td>Miltenyi Biotec</td>
<td>&nbsp;</td>
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detection and isolation of viable mouse il-17-secreting t cells

La secrezione delle citochine MACS tecnologia di analisi permette di individuare i citochine secrete a livello di singola cellula e l&#39;isolamento sensibile di valide cellule che secernono citochine. Al fine di etichetta IL-17-cellule che secernono, una sospensione singola cella di splenociti mouse è preparato e stimolato a 37 ° C con PMA / ionomicina per indurre la secrezione di citochine. Per arrestare la secrezione delle cellule vengono poi immessi sul ghiaccio e sono esposti al IL-17 reagente Catch a bi-anticorpo specifico che si lega al CD45 sulla superficie cellulare dei leucociti e di IL-17 come è secreta e catturato vicino alla superficie della cellula. Secrezione è poi ri-iniziata aumentando la temperatura a 37 ° C e IL-17 è intrappolato dal reagente Catch. Secrezione è poi fermato di nuovo, mettendo cellule su ghiaccio. Per rilevare l&#39;IL-17 intrappolati, le cellule vengono incubate con un secondo IL-17-anticorpo specifico coniugato con biotina e un anti-biotina-PE anticorpi. Le cellule possono ora essere analizzati direttamente mediante citometria di flusso o preparati per l&#39;isolamento e l&#39;arricchimento per l&#39;etichettatura successive con Anti-PE microsfere coniugate.

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

Questa procedura descrive l&#39;individuazione e isolamento di mouse leucociti TH17 che attivamente secernono IL-17 sulla stimolazione.

Rilevazione e isolamento delle vitali del mouse IL-17-cellule che secernono 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 Views.  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.

Isolamento di cellule T CD4 + da Linfonodi mouse utilizzando Miltenyi Purificazione 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 ...

Ricerca

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.

L&#39;isolamento e la derivazione di cellule embrionali del mouse 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 di cellule endoteliali valvolare

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 di cellule staminali del mouse ghiandole salivari

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 delle cellule basali e sottomucosa cellule del dotto di Gland 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. ...

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 del mouse principale acinose pancreatiche Cellule

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.

Isolamento e colture primarie di cellule di topo aortica endoteliali

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 di cellule di topo coronarica endoteliali

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.