Reaktifler hazırlanması <ol><li> BSA (% 0.5) ve 2 mM EDTA içeren PBS ile bir tampon yapın. MACS ayırma kolonları hava kabarcıklarını engellemek için, 2-8 ° C Kullanmadan önce tampon gazlar ve saklanacak. </li><li> Biz RPMI 1640 orta% 5 fare içeren serum kullanın. Kültür ortamı, bu bileşiklerin hücre stimülasyon özgüllüğü değiştirmek gibi, BSA veya FCS içermemelidir. </li><li> Fare IL-17 sekresyonu Testi - Miltenyi-Biotec Hücre Zenginleştirme ve Tespit Kiti. IL-17 Yakalama Reaktif, IL-17 Algılama Antikor (biotin), Anti-Biotin-PE ve Anti-PE mikro kiti aşağıdaki bileşenleri içerir. </li></ol> Splenositlerin Uyarıcı <ol><li> Bu protokol unstimulated dalak ve T hücreleri için bir karşı negatif ve pozitif kontrol, huzurunda yapılır. Bu protokol, steril tekniği kullanılarak yürütülmektedir. </li><li> GentleMACS ™ Dissociator kullanılarak izole edildi fare dalak tek bir hücre süspansiyonu hazırlayın. Hücre konsantrasyonu hücre sayımı ile önceden belirlenmiş olmalıdır. Pelet oda sıcaklığında 10 dakika için 200 × g hücreleri. </li><li> Santrifüj sonrasında, bir pipet kullanarak pelet kapalı supernatant aspirat. Pelet kaybını önlemek için tüp süzün. </li><li> Şimdi, kültür ortamı hücreleri tekrar süspansiyon haline getirin ve sonra iyi bir eklemek. ML başına on milyon hücreleri ve santimetrekare başına beş milyon hücre bir konsantrasyon için yeterli orta ekleyin. </li><li> Yeniden süspanse hücreleri bağışıklık yanıtının teşvik etmek, örnek ionomycin (1 mcg / ml) ve PMA (10 ng / ml) ekleyin ve karışımı hafifçe yukarı ve aşağı pipetleme çözüm. Daha sonra kuyu buna göre etiketlenir. </li><li> Şimdi, bizim hücreleri 3 saat için 37 ° inkübe olacak ° C uyarılması dönemi başlatmak için herhangi bir karışım. Stimülasyon başlangıcından IL-17 analizi sonucunda 3 saat devam edin, buna göre planlayın. </li><li> Salgılanması durdurmak için, hücre buz üzerine yerleştirilir ve soğuk tampon yavaşça yukarı ve aşağı pipetleme tarafından uyarılmış hücreleri toplamak. Hücreler daha sonra iyi bir tüpe aktarılır ve ikinci kez yıkanır. </li><li> Tüm hücreleri toplanır emin olmak için, bir mikroskop altında çanak kontrol etmek için iyi bir fikirdir. Eğer hücreler hala bağlı kalır, soğuk tamponu ile bulaşık durulama kalan hücreleri toplamak. Herhangi bir hücre kümeleri hücre süspansiyonu öncesi ayırma filtreleri kullanarak silinebilir. </li></ol> Yakalama Reaktif Hücreler Etiketleme <ol><li> IL-17 salgılayan hücrelerin% 2&#39;den az bulunması halinde bu testte en iyi şekilde çalışıp çalışmadığını, dikkat etmek çok önemlidir. </li><li> , IL-17 salgılayan hücreleri konsantrasyonu, buna göre hacimleri ayarla% 2 daha fazla olması bekleniyor. <img src="/files/ftp_upload/old/1037_2008_12_8_6/figure8.png" alt="Şekil 8" width="500" height="162" /></li><li> Bu prosedürün potansiyel Bir hatadır Catch Reaktif çapraz kontaminasyon, iki spesifik antikor böylece yanlış pozitif olmayan bir salgılayan T hücre ve tuzakları bir komşu lenfosit salgılanan IL-17 üreten, bağlanır etiketleme sırasında oluşabilir. </li><li> Bu sorunu aşmak için, IL-17 difüzyon yavaş ve bu çapraz bulaşmayı önlemek için önce soğuk tamponu ile etiketleme ve iş aşağı soğuk hücreler için kritik öneme sahiptir. Hücreleri soğuk tutmak için ek olarak, belirli bir konsantrasyon tutulmalıdır. </li><li> Etiketleme prosedürü başlatmak için, 15 ml kapaklı tüp içinde on milyon hücreleri kullanır. Kullanılmak üzere yüksek hücre sayıları varsa, sadece buna bağlı tüm birimler büyütmek. Optimal hücre konsantrasyonu elde edildikten sonra, 10 ml soğuk tampon ekleyerek hücreleri yıkayın. </li><li> Soğutmalı santrifüj (2-8 ° C) 10 dakika için 300 hücreleri × g dönerler. </li><li> Santrifüj sonra, tamamen bir pipet kullanarak süpernatant aspirat. Bu hücre kaybı ve kesin olmayan birimler yol açacak gibi süpernatant süzün. Soğuk tampon, santrifüj ve aspirasyon 10ml ekleyerek oluşan yıkama adımı yineleyin. </li><li> Şimdi biz bir pelet istenilen saflık olduğunu, soğuk kültür ortamı 80 mcL hücreleri tekrar süspansiyon haline getirin. Etiketlemek için, şimdi 20 mcL Fare IL-17 Catch Reaktif katacak. </li><li> 5 dakika buz üzerinde hücrelerin inkübe edin. </li><li> 5 dakika buz üzerinde kuluçka döneminden sonra, tüp kaldırmak ve 37 ° C sıcak bir ortamda 10 ml hücrelerin sulandırmak. Sonra MACSmix Tüp Rotator tüp güvenli ve tüp inkübe 37 ° C altında 45 dakika süreyle sürekli hareket. Sıcaklığının artmasıyla 37 ° C sitokin salgılanması yeniden başlayacaktır. </li></ol> IL-17 Algılama Antikor (Biotin) ve Anti-Biotin-PE Hücreler Etiketleme <ol><li> 37 ° C&#39;de 45 dakika salgılanması süre sonra, buz üzerinde hemen tüp yerleştirin. Bu sitokin salgılanması durur. Burada üzerinde buz üzerinde hücre tutmak için kritik öneme sahiptir.Soğuk tampon Çalışma Catch Reaktif çapraz kontaminasyonu engellemek. </li><li> 2-8 300xg soğuk tampon ve santrifüj ° C de 10 dakika ile tüp doldurun. Süpernatantı tamamen aspire. Yıkama, bir kez daha adımı yineleyin. </li><li> Hücre pelletini soğuk tampon 80 mcL yeniden süspanse ve buz üzerinde tüp yerleştirilir. </li><li> Antikor belirsiz bağlanma önlemek için, 5 dakika buz 10μl FCR Engelleme Reaktif ve inkübe eklenmesi önerilir. Sonra, 10 dakika boyunca buz üzerinde biotin ve inkübe konjuge Fare IL-17 Algılama Antikor (Biotin) 20 mcL ekleyin. </li><li> Bir kez daha, 2-8 300xg soğuk tampon ve santrifüj 10 ml ekleyerek ° C 10 dakika önce yaptığı gibi hücrelerin yıkayın. </li><li> Şimdi süpernatant aspirat ve soğuk tampon 80 ul hücre pelletini tekrar süspansiyon haline getirin. </li><li> Ikincil antikor, Anti-Biotin-PE 20 mcL ekleyin. </li><li> Hücreler ve sekonder antikor çözüm karıştırın ve 10 dakika boyunca buz üzerinde tüp tekrar kuluçkaya yatmaktadır. </li><li> Bu ikinci inkübasyon tamamlandıktan sonra, 10 ml soğuk tampon ve santrifüj ile hücreleri yıkayın. </li><li> Pelet şimdi soğuk tampon 500 mcL yeniden süspanse edilebilir. </li><li> Hücreler daha fazla alt analizi için ayrılacak varsa, bunlar manyetik Anti-PE-mikro etiketli ve MACS Sütunlar ve MACS Ayırıcılar kullanarak elle ya da otomatik ayrılmış olabilir. </li><li> Şimdi splenositlerin etiketleme ve analiz için hazır hale gelmiş. </li></ol> FACS Analizi <ol><li> Bizim analizi için, flow sitometri ile uyarılır ve unstimulated splenositlerin karşılaştırabilirsiniz. </li><li> Akım sitometri analizi önce, hücrelerin ölü hücreleri çıkış kapısı propidium iyodür ile 0.5 mg / ml lekeli. MACSQuant ™ Analyzer, her bir örnek için 200.000 hücre yüklü ve şimdi örnekleri göreli antikor reaktivite dağılım araziler bakmaya devam etmektedir. Nadir hücrelerinin analizi için iki önemli hususlar - IL-17 pozitif hücrelerin gibi flow sitometri ile: <ul><li> forward scatter karşı yan scatter plot lenfosit bir kapı ayarı </li><li> ve ölü hücreleri (propidium iyodür ile boyanmış) ve B-hücrelerinin (belirsiz arka plan boyama yol açabilir) dışarı yolluk algılama hassasiyeti daha da geliştirmek için </li></ul></li><li> , CD4-APC T hücreleri ve B hücreleri algılamak için CD45R/B220-PerCP antikor tespit antikor kullanılır. Biz örneklerin ileri ve yan dağılım özelliklerini görmek ve lenfosit nüfus üzerinde bir kapı uygulamak. </li><li> Lekeli ölü hücreleri ve lekeli B hücreleri (Y ekseni) görmek için ikinci bir kapı yapıldı. Bu hücreler, T hücre analizi hariç tutulur. </li><li> Bu örnekte, aslında bizim negatif kontrol, biz unstimulated örnekleri çok az sayıda IL-17 salgılayan CD4 pozitif T hücreleri olduğunu görebilirsiniz - yaklaşık% 0.003 (Şekil 4). Uyarılmış T hücre popülasyonu baktığımızda, önemli sayıda CD4 pozitif T hücreleri görebilirsiniz salgılar IL-17 - ayırma önce 0,367% (Şekil 5A) ve MACS Teknoloji (Şekil 5B) ile manyetik ayırma sonra% 60.76. </li></ol>

Tüm protokolleri ve veri sayfaları www.miltenyibiotec.com mevcuttur. Uyarılar Reaktifler sodyum azit içerir. Asidik şartlar altında, son derece toksik olan hidrazoik asit, sodyum azid verir. Atma atılmadan önce akan su ile seyreltilmiş olmalıdır. Bu önlemler patlayıcı koşullarının daha da geliştirilmesi sıhhi tesisat birikmeleri önlemek için tavsiye edilir.

Th17 hücreleri adaptif bağışıklık ve inflamatuvar yanıt tamamlayıcı bir rol oynamaktadır ve otoimmun inflamasyonu sürüş önemli bir bileşenidir. Bu video Miltenyi Kullanıcı Fare nasıl kullanılacağını belgeler IL-17 sekresyonu Testi - Hücre Zenginleştirme ve Tespit Kiti (PE). Bu işlemi yaparken, IL-17 salgılayan hücrelerin başlangıç ​​hücre hazırlama frekans bir tahmin olması önemlidir. Etiketleme ve sitokin sekresyon dönemi sırasında uygun hücre konsantrasyonu süspansiyon diğer hücreler tarafından çapraz bulaşmayı önler ve güvenilir sonuçlar sağlar.

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

MACS Sitokin Salgı Testi teknolojisi, tek hücre düzeyinde ve yaşayabilir sitokin salgılayan hücreleri hassas izolasyon salgılanan sitokinlerin algılanmasını sağlar. IL-17 salgılayan hücreleri, fare dalak tek bir hücre süspansiyonu ° C PMA / ionomycin sitokin salgılanması neden 37 hazırlanan ve uyarılır etiketlemek için. Sekresyonunu hücreleri durdurmak için bir iki spesifik antikor salgılanır ve hücre yüzeyine yakın yakalandı olarak IL-17 ve lökositlerin hücre yüzeyinde CD45 bağlanır, sonra buz üzerine yerleştirilir ve IL-17 Yakalama Reaktif maruz kalmaktadır. Salgı sonra sıcaklık 37 artırarak yeniden başladı ° C ve IL-17 Catch Reaktif tarafından tutulur. Salgı sonra buz üzerinde hücreleri yerleştirerek, tekrar durdurulur. Kapana kısılmış bir IL-17 algılamak için, hücreler, biotin için konjuge ikinci bir IL-17-spesifik antikor ve Anti-Biotin-PE antikor ile inkübe edilir. Hücreler artık doğrudan akış sitometri ile analiz veya Anti-PE konjuge mikro sonraki etiketleme izolasyon ve zenginleşmesi için hazırlanmış olabilir.

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

Bu prosedür, tespiti ve izolasyonu stimülasyon üzerine IL-17 aktif salgılar fare Th17 lökositlerin açıklamaktadır.

Canlı Fare IL-17 salgılayan T Hücreler Algılama ve İzolasyonu

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

Research

JoVE Journal

Biology

11.1K Görüntüleme.  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.

CD4 İzolasyon Miltenyi MACS Arıtma Fare lenf düğümleri + T hücreleri

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

Biyoloji

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.

Fare Embriyonik Germinal Hücreleri İzolasyon ve türetilmesi

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.

Kapak Endotel Hücreleri İzolasyon

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.

Fare Tükürük Bezi Kök Hücre İzolasyonu

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.

Fare Trakea gelen Bazal Hücre ve Submukozal Bezi Kanal Hücre İzolasyonu

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.

Fare Primer Pankreas Asiner Hücreleri İzolasyon ve Kültür

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.

İzolasyon ve Fare aort endotel hücreleri İlköğretim Kültür

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.

Fare Koroner Endotel Hücreleri İzolasyon

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.