The authors thank Sanjiv Luther and colleagues for helpful discussions in establishing the current lymph node digestion protocol. This work was supported by SNF grants PPOOA-_119204 and PPOOP3_144918 to S.W.R.

Bu görüntü yayını ve protokol, tüm hayvan prosedürleri Kanton Kurumu, Basel-Stadt, İsviçre tarafından onaylanan hayvan protokole uygun olarak yapılmıştır. 1. Lenf düğümleri Hazırlama ve Sindirim <ol><li> Isıtma plakası ile bir manyetik karıştırıcı üzerinde 37 ° C bir cam kapta Ön-ısıtma su. </li><li> Aşağıdaki gibi temel Orta hazırlayın: DMEM ortamı (piruvat olmadan),% 2 FCS ile takviye edilmiş, 1.2 mM CaCl2 ve pen / strep (penisilin 100 birim, streptomisin, 100 ug). </li><li> Kullanmadan önce tüm diseksiyon aletleri sterilize edin. </li><li> CO 2 boğulma başına lenf nodu donör fareler Euthanize ve aseptik lenf düğümleri teşrih. Çevreleyen yağ teşrih etmeyin. NOT: Bu protokol periferik cilt-lenf düğümünde (inguinal, brakiyal, aksiller) için optimize edilmiştir. </li><li> 2 ml buz soğukluğunda bazik ortam ihtiva eden steril bir Petri kabı, lenf düğümleri yerleştirin. </li><li> Lenf nodu ca Disrupt1 ml şırınga sabit iki 25 G iğne kullanılarak psule. </li><li> 1 mg / ml kollajenaz IV, 40 ug / ml DNAz I ile takviye edilmiş 750 ul Temel ortamı içeren 5 ml&#39;lik bir polipropilen yuvarlak dipli tüp içinde kesintiye lenf nodu doku aktarın </li><li> Her bir tüp bir manyetik karıştırıcı, steril ekleyin. </li><li> 37 ° C ile çanak içinde yer tüpün artık su ısıtılır ve 30 dakika boyunca yavaş bir hızda (1 tur / sec) tüplerini karıştırın. </li><li> Isıtma plakası ile manyetik karıştırıcı gelen tüp çıkarın ve lenf nodu fragmanları halledelim. </li><li> Dikkatle &quot;non-stromal hücre&quot; zenginleştirilmiş süpernatant kaldırmak. NOT: T, B, dendritik hücreler ve CD45 analizi ise - gp38 - CD31 - öngörülen, &quot;non-stromal hücreler&quot; kısmını kaydedin. </li><li> Yıkama 750 ul ortam maddesi ile bazik lenf düğümü dokusu kalmıştır. NOT: Bu adım, bağışıklık yetkin fareler ile çalışan sadece gereklidir. </li><li> Lenf nodu fragmanları halledelim. </li><li> Kaldırolmayan stromal hücre-yüzen fraksiyonu. </li><li> 750 ul temel orta 3,5 mg ile takviye lenf nodu fragmanları Ekle / ml kollajenaz D ve 40 ug / ml Dnase I. </li><li> Lütfen 37 ° C de içeren beher içinde yer tüpün artık su ısıtılır. </li><li> Yavaş yavaş karıştırılırken buna 5 dakika boyunca lenf nodu dokusu Digest. </li><li> Pipetleme ve çok kanallı otomatik pipet kullanılarak maksimal hızda, 10 döngü boyunca 700 ul karıştırılarak Ayrık lemf nod dokusu parçalan olabilir. NOT: Bu sindirim geliştirmek için lenf nodu doku bozar. </li><li> 37 ° C&#39;ye ısıtılmış su ile tekrar beher tüpü yerleştirin. </li><li> Yavaş yavaş karıştırılırken buna 10 dakika daha Özet lenf nodu dokusu parçalan olabilir. </li><li> Pipetleme lemf nod dokusu parçaları parçalamak ve bir çok kanallı otomatik pipet kullanılarak maksimal hızda, 99 çevrim için karıştırılması. </li><li> Tek bir hücre süspansiyonu bakımı için, 0.5 M EDTA, 7.5 ul ekleyin. </li><li> Boru itibariyle ayrılmış lemf nod dokusu parçalantting ve çok kanallı otomatik pipet kullanılarak maksimal hızda, 99 çevrim için karıştırılması. </li><li> 750 ul temel orta ekleyin ve 70 mikron naylon örgü ile hücrelere geçerler. </li><li> Santrifüj Hücre süspansiyonu 1500 xg&#39;de 5 dakika, 4 ° C. </li><li> Stromal hücreler, bundan başka analizler için de kullanılabilir. </li></ol> Lenf Düğümü Stromal Hücrelerinin 2. Boyama <ol><li> Başarılı bir şekilde TRC LEC BEC DN hücreleri tanıyan anti-CD45, anti-podoplanin (gp38), anti-CD31 antikorlarının bir kombinasyonu kullanılır. </li><li> Anti-gp38-PE (1: 200), anti-CD31-APC (1: 200), 100 ul HBSS içinde 2 ihtiva eden: Canlı / Ölü izleyici, bir anti-CD45-FITC (200 1) ile sindirilmiş lenf düğümü dokusu inkübe karanlıkta en az 20 dakika, 4 ° C,% FCS için. </li><li> % 2 FCS ihtiva eden HBSS 500 eklenmesinin Hücreleri yıkamak </li><li> Santrifüj Hücre süspansiyonu 1500 xg&#39;de 3 dakika, 4 ° C. </li><li> % 2 FCS içeren HBSS 100 ul yeniden askıya. </li><li> Equipp bir akış sitometresinde boyalı hücrelerin çalıştırınAşağıdaki optik ile ed: uyarma kadar üç lazer ile kaynak: mavi (488 nm, hava soğutmalı, 20 mW katı hal), kırmızı (633 nm, 17 mW HeNe), mor (405 nm, 30 mW katı hal) . </li><li> Kapısı CD45 - hücreler hematopoietik hücreleri hariç. </li><li> Kapısı mayoları (FSC-W) ve canlı hücreleri (CANLI / DEAD izci) ikilileri ve ölü hücreleri hariç. </li><li> , LEC (gp38 + CD31 +), BEC (gp38 - CD31 +) ve DN (gp38 - CD31 -) hücreleri (Şekil 1) - CD31 vs Arsa gp38 TRC (gp38 + CD31) görselleştirmek için. </li></ol>

<ol>
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The authors declare they have no competing financial interests.

Lenf nodu stromal hücrelerin çalışması nedeniyle son zamanlarda yayımlanmış iki sindirim protokolleri 6,13 gelişimine bir araştırma odağı haline geldi. Her iki protokol tek bir lenf düğümü stromal hücreler elde etmek için yeterli, ancak sindirim enzimlerinin kullanılması ve sindirim zaman içinde farklılık gösterir. Stromal hücreler ve onların yüzey belirteçleri enzimatik sindirim ve mekanik strese karşı hassas olduğu için, optimize edilmiş bir protokol gereklidir. <p clas...

Lenf düğümleri, yabancı ve öz-antijenlere karşı adaptif immün yanıtları başlatılan ve koordine özelleşmiş bölmeleri bulunmaktadır. Burada sunulan prosedür lenf düğümü, tek bir hücre süspansiyonu elde etmek ve çeşitli moleküllerinin yüzey ekspresyonunu korumak uygun bir lenf düğümü stromal hücrelerin erişmek için kullanılan otomatik mekanik pipetleme ile birlikte kısa bir enzimatik sindirim açıklanmaktadır. Lenf nodu stromal hücre üç önemli fonksiyonları lenf nodu iskele oluşturacak ve yerine: ilk onlar vücut sıvıları antijenleri, patojenleri ve onların patojen ilişkili moleküler pattern (PAMP) yanı sıra, sitokinler ve tehlike ilişkili moleküler model örnek filtre (Damps) mevcut Vücutta. İkincisi, etkileşim ve adaptif immün yanıtları başlatmak için antijen sunan hücreler (APC) ve lenfositler çekmek ve talimat; ve üçüncüsü, lenfosit 1 homeostası farklılaşması için yapısal bir ortam sağlamak-3. Inflamasyon lenf düğümü, stromal hücreleri büyüme faktörleri, sitokinler ve kemokinlerin üretimi sırasında, dendritik hücreler (DCler) arasındaki etkileşimi düzenlemek ve böylece, T-ve B-hücrelerinin adapte şişme. Bağışıklık tepkilerinin düzenleme, farklı stromal hücre popülasyonları tarafından meydana getirilen kompleks yapısal mimarisine mümkündür. Lenf nodülü hücreleri, stromal, CD45 negatif hücrelerdir ve fibroblastik ve endotel hücreleri 1 -6 &#39;de CD31 ile gp38 ifadesi ya da ayırt edilebilir. CD31 + kan endotel hücreleri tanımlar (BEC) -, gp38 + CD31 + lenfatik endotel hücreleri (LIK), gp38 tanımlar: - Gp38 + CD31 (fibroblastik retikulum hücreleri de FRC olarak bilinen TRC) T bölgesi retiküler hücreleri tanımlar. Bundan başka, alt-popülasyonlarının tanımlanması diğer lenf nodu stromal hücrelerin varlığını ortaya çıkarmıştır. Gerçekten de, küçük bir perisit benzeri hücre popülasyonu içinde karakterize edilmiştirgp38 - CD31 - nüfus 7. Bu nedenle, izolasyon prosedürü adaptasyonu farklı lenf düğümü stromal hücrelerin fonksiyonel özelliklerinin tanımlanması ve karakterizasyonu için avantajlıdır. Lenf düğümü hücreleri, stromal sindirim geliştirilmesi önce lenf düğümü stromal hücrelerin doku çalışma bölümü ve mikroskopi kullanılarak yerinde gözlemlerle sınırlıydı protokolleri. Bununla birlikte, yapısal ve fonksiyonel çalışmalar lenf düğümü stromal hücrelerin önemli özellikleri gösterdi. Lenf nodülü hücreleri, stromal yüksek endotelial lenf nodu subkapsüler sinüs lenf ve ilgili-düşük molekül kütlesi, protein nakil kanal sistemi olarak adlandırılan karmaşık bir 3 boyutlu bir yapı oluşturmak üzere podoplanin, kolajen ve hücre dışı matris (ECM), proteinleri ile ilişkili T hücreleri bölgesinde 8 Venüller. DCler, stroma hücreleri ile yakın temas halinde olan ve boru şeklindeki con içine uzanan gözlemlenebilirDuit yapısı sıvı örneklemek ve antijenleri 8 algılamak için. DCler ile lenf düğümü stromal hücreler (Kızılay ve LECS) etkileşimi, kemokinler CCL21 ve CCL19 9,10 serbest bırakılması ve sunum aracılık eder. CCL19 ve CCL21 lenf nodu T hücre bölgesine 4,11 göç etmek DH&#39;leri ve T hücrelerini kolaylaştırılması CCR7 reseptörü tarafından tanınır. Benzer kemokinlerle kullanarak rağmen, DC&#39;ler ve T hücreleri lenf düğümlerine 12 içine farklı göç yolları var. Daha sonra, lenf düğümü ve saf lenf düğümü stromal hücrelerin izolasyonu enzimatik sindirim kullanılarak, fonksiyonel çalışmalar çeşitli lenf nodu stromal hücrelerin rolü DC&#39;ler ve T / B hücreleri 6,13 ile etkileşim yetenekleri üzerinde gerçekleştirilmiştir. İlk olarak, IFN-γ üreten efektör T hücrelerinin ve lenf düğümü stromal hücreler arasındaki karışma, ikincil lenfoid organların 14-16 T hücre yanıtları ve çoğalmasını azaltmak için gösterilen metabolit nitrik oksit üretimini indükler. İkinci olarak, lenf Kgazel stromal hücreler, IL-10, 17 üretimi ile düzenleyici DC alt kümelerinden farklılaşmasını desteklemek için ve IL-7 6,18 üretilmesi yoluyla saf T hücre homeostazının modüle bildirilmiştir. Üçüncü olarak, lenf düğümü stromal hücrelerde TLR sentezleme stromal hücreler, doku yaralanması sırasında ortaya çıkan bir enfeksiyon ya da kendi kendini moleküllerden türetilmektedir sinyale duyarlı olduğunu göstermektedir. Gerçekten de, TLR3 poli ligandla lenf düğümü stromal hücrelerin tedavisi (I: C) ile sonuçlanan büyük histo-uyumluluk kompleksi sınıf I ve eş-ifade önleyici molekülün PD-L1 upregülasyonu mütevazı bir düzenlenişini uyarmaktadır değil, birlikte uyaran moleküllerin periferal dokuda dramatik değişiklikler ifadesini 19 antijenleri. Çeşitli gruplar, lenf düğümü, stromal hücreleri periferal doku antijenleri ifade eder ve kendinden reaktif T hücrelerinin 19,21-27 indüklenmesidir göstermiştir. Bu nedenle, lenf düğümü hücreleri ve diğer göçmen ve yeniden arasındaki etkileşimleri anlamaksident lenf düğümü hücreleri iltihap sırasında aktivasyonu ya da bağışıklık tepkilerinin bastırılması sağlamak için yeni bir hedef moleküller bulmak için yardımcı olacaktır. Bu nedenle, lenf düğümü yayınlanmış enzimatik ayırma uygulanması gereklidir. Daha önce yayınlanmış protokoller düşük mekanik stres 6,19,20 ile kollajenaz tabanlı enzimatik sindirim farklı kombinasyonları kullanın. Bununla birlikte, sindirim enzimlerinin veya sindirim enzim farklı bir kombinasyonu ile uzun inkubasyon aktivasyon durumunu analiz etmek ve yeni lenf nodu stromal hücreleri tanımlamak için gereken çeşitli yüzey molekülleri düşürebilir. Stromal hücre analiz türüne bağlı olarak, bağlantı veya Protokol Protokol Fletcher daha uygun olabilir. Tarif edilen prosedürde, biraz daha kısa bir enzimatik sindirim uygun bir lenf düğümü stromal hücrelerin yüzey işaretleyici zararın en aza düşürülmesi için otomatik mekanik ayrıştırma ile bir araya getirilmektedir. Bu işlem, yüksek ölçüde tekrarlanabilir ayrılmasını sağlayan vedüşük değişkenlik ve% 95 daha fazla canlılığı ile lenf nodu stromal hücre popülasyonlarının ayrım. Taze olarak izole edilen lenf nodülü hücreleri, stromal, doğrudan in vitro fonksiyonel deneyleri gerçekleştirmek için, stromal hücreleri hatlarının yüzey markeri ekspresyonu, protein analizi ve transkripsiyon çalışmalar, hem de kurulması için kullanılabilir.

<table><tbody><tr><td>DMEM</td><td>LifeTechnologies-Gibco</td><td>41965-039</td><td></td></tr><tr><td>FCS</td><td></td><td></td><td>Final concentration 2%</td></tr><tr><td>CaCl2</td><td>Sigma</td><td>499609</td><td>Final concentration 1.2 mM</td></tr><tr><td>Collagenase IV</td><td>Worthington Biochemical Corporation</td><td></td><td>&gt;160 units per mg dry weight, use at final concentration of 1 mg/ml</td></tr><tr><td>Collagenase D</td><td>Roche</td><td>11088882001</td><td>use at 3.5 mg/ml</td></tr><tr><td>DNAse I</td><td>Roche&amp;nbsp;</td><td>11284932001</td><td>use at 40 &amp;micro;g/ml</td></tr><tr><td>Stiring magnets</td><td>FAUST</td><td></td><td>5 mm long-2 mm &amp;oslash;</td></tr><tr><td>Polystyrene round bottom tubes 5 ml</td><td>Falcon-BD Bioscience</td><td></td><td></td></tr><tr><td>Magnetic stirrer with heating function</td><td>IKA-RCT-standard</td><td>9720250</td><td></td></tr><tr><td>Petri dishes 100 mm, sterile</td><td>TPP</td><td>6223201</td><td></td></tr><tr><td>25 G needles</td><td>Terumo</td><td></td><td></td></tr><tr><td>anti mouse CD45 Ab</td><td>Biolegend</td><td></td><td>Clone 30-F11</td></tr><tr><td>anti mouse CD11c Ab</td><td>Biolegend</td><td></td><td>Clone N418</td></tr><tr><td>anti mouse Podoplanin Ab</td><td>Biolegend</td><td></td><td>Clone 8.1.1</td></tr><tr><td>anti mouse CD31 Ab</td><td>Biolegend</td><td></td><td>Clone MEC13.3</td></tr><tr><td>&lt;br/&gt; Eppendorf Xplorer plus, Multichannel</td><td>Eppendorf</td><td>4861 000.821/830</td><td>1.250 &amp;micro;l max. volume</td></tr><tr><td>anti mouse CD140a</td><td>Biolegend</td><td></td><td>Clone APA5</td></tr><tr><td>anti mouse CD80</td><td>Biolegend</td><td></td><td>Clone 16-10A1</td></tr><tr><td>anti mouse CD40</td><td>Biolegend</td><td></td><td>Clone 1C10</td></tr><tr><td>anti mouse I-Ab</td><td>Biolegend</td><td></td><td>Clone AF6-120.1</td></tr><tr><td>anti mouse CD274 (PD-L1)</td><td>Biolegend</td><td></td><td>Clone 10F.9G2</td></tr><tr><td>LIVE/DEAD Fixable Near-IR Dead Cell stain kit</td><td>Invitrogen</td><td>L10119</td><td></td></tr></tbody></table>

isolation of murine lymph node stromal cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Mevcut protokol nedeniyle bir çok kanallı otomatik pipet ile mekanik çözümlemesiyle kısa bir sindirim süre (45 dk maksimum) ile Bağlantı ve ark., 6, 2007 tarafından yayınlanan bir modifiye sindirim protokoldür. Buna ek olarak, işlem, daha standart, farklı lenf düğümü stromal hücrelerin yüzey markerlerinin bozulmasını en aza indiren ve aynı anda birden fazla numunenin kullanılması mümkün olur. Collagenasein IV ve Kollagenaz D Fletchers protokolü...

Watch this Scientific Journal Video about Isolation of Murine Lymph Node Stromal Cells at JoVE.com

Isolation of Murine Lymph Node Stromal Cells

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Kemirgen Lenf Düğümü Stromal Hücrelerinin İzolasyonu

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

isolation-of-murine-lymph-node-stromal-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

31.1K Görüntüleme.  University of Basel and University Hospital Basel. Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells. 

Video: Isolation of Murine Lymph Node Stromal 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 of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Fare Genital Sistemlerinin Mukozanin gelen Lenfosit İzolasyonu

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

İmmünoloji ve Enfeksiyon

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

İzolasyon ve naif CD4 T Lenfositler Th17 Farklılaşma

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

Lenf düğümü Stromal Hücre Menşe Çalışmaları Lenf-yağ Pad Kimeralar Üretimi

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

İzolasyon, tanımlama ve Murin Thymic Epitel Hücreleri saflaştırılması

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

Fare Küçük Bağırsak Lenfositler izolasyonu ve Akım Sitometrisi Karakterizasyonu

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

İzolasyon ve murin lenfositlerin aktivasyonu

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Lenfositler fare küçük bağırsak bağışıklık sistemi yalıtma

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Timus ve Kemik İliğinden Stromal Hücre İzolasyonunun Optimize Edilmesi

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

Yazar Spotlight: Tek Hücre Dizilimi için Stromal Hücre İzolasyonu Kullanarak Hematopoietik Araştırmaları İlerletmek

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

Hematopoetik Organlardan Stromal Hücre İzolasyonu

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

Kemirgen Lenf Düğümü Stromal Hücrelerinin İzolasyonu

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

CD4 İzolasyon Miltenyi MACS Arıtma Fare lenf düğümleri + T hücreleri

Fare Genital Sistemlerinin Mukozanin gelen Lenfosit İzolasyonu

İzolasyon ve naif CD4 T Lenfositler Th17 Farklılaşma

Lenf düğümü Stromal Hücre Menşe Çalışmaları Lenf-yağ Pad Kimeralar Üretimi

İzolasyon, tanımlama ve Murin Thymic Epitel Hücreleri saflaştırılması

Fare Küçük Bağırsak Lenfositler izolasyonu ve Akım Sitometrisi Karakterizasyonu

İzolasyon ve murin lenfositlerin aktivasyonu

Lenfositler fare küçük bağırsak bağışıklık sistemi yalıtma

Hematopoetik Organlardan Stromal Hücre İzolasyonu

Hematopoetik Organlardan Stromal Hücre İzolasyonu