1。三次元共培養を確立する<ol><li>共培養を確立する前に、ある日、一晩4℃で冷蔵庫で氷の上で冷凍庫で、解凍からマトリゲルを削除します。 </li><li>実験の日に、使用する上皮細胞の培地に対応する共培養のため培地を準備します（この例ではHMEC培地：10％仔ウシ血清、5μg/ mLのインスリンDME/F12培地1μg/ mLのヒドロコルチゾン、および1 ng / mLのEGF）。マトリゲルと混合する前に氷の少なくとも1時間で培地を保持します。 </li><li>解凍したマトリゲルを希釈し、マトリゲルの合計所望の量を決定します。培地（24ウェルプレートのウェルあたり300μL）と氷上に滅菌チューブに氷冷したHMEC培地の半分をそのボリュームを追加します。その後1:1希釈を達成するためにチューブに解凍マトリゲルの同じハーフの総ボリュームを追加します。チューブを氷上に保持します。 </li><li>場所50マトリゲルのμL：24ウェルプレートのウェル内の指定されたミッドウェル培地混合物と加湿空気および5％CO 2、37℃インキュベーター内で10分間インキュベートする。 </li><li>ウェルに培地混合物と別の60分間インキュベート：マトリゲルの追加450μLを追加します。 </li><li>インキュベーション中に、0.5×10の濃度で繊維芽細胞（ここでGFPを発現するH-tert-不死削減乳腺線維芽細胞）および上皮細胞（ここではRFPを発現しているH-tert-不死化ヒト乳腺上皮細胞株）の1:1懸濁液を調製HMEC培地0.5mlに5個の細胞それぞれ。 </li><li> 60分のインキュベーションステップの後、静かに固化したゲルの一番上に細胞懸濁液をドロップし、インキュベーターに文化を返します。 </li><li> 2日間培養を乱すことがありません。この後、培地を非常にゆっくりと井戸の側から培地を吸引し、優しく新鮮なHMEC培地で置き換えることにより、2〜3日ごとに変更することができます。 </li><li>顕微鏡下で毎日のスフェロイド形成を監視することができます。文化は10-14日後に一般的に成熟している。イメージとして光や蛍光顕微鏡を使用して、適切な。回転楕円体のサイズと投影の長さは光学顕微鏡下で測定することができます。 </li></ol> 2。共培養スフェロイドの細胞の分離<ol><li>非常に穏やかに、文化をピペットと滅菌ダウン2 mLのガラスピペットを用いて10倍。また、文化をピペットするために、70％エタノールでオートクレーブまたは浸漬により滅菌はさみを使用する1000マイクロリットルプラスチック製のピペットチップの先端を切り落とした。ガラスピペットまたは滅菌15 ml遠心チューブにカットプラスチックピペットチップと室温で2000rpmで5分間遠心分離のいずれかで文化を転送します。携帯スフェロイドは、堆積物下部になります。 </li><li>軽く遠心分離管の上部から中断マトリゲルを削除するには、滅菌パスツールピペットの先端を使用しています。 </li><li> 、ペレット化したスフェロイドを1ミリリットルHMEC媒体を追加します。滅菌2mLのガラスピペットで2〜3回ピペッティングし、24ウェルのウェルに文化を転送組織培養プレート。 </li><li>スフェロイドは、媒体を変更する前に少なくとも48時間は皿にアタッチすることができます。時間の経過とともに細胞が球状構造を残し、単層として成長します。 </li><li>通過培養細胞を細胞が50％コンフルエントに達する。最初の35 mmディッシュ（約2-4日）に、その後100 mmディッシュ（約3-5日）に文化を展開します。各通過ステップの直前に、手動で可能な限り多くの残留 &quot;ゴーストスフェロイド&quot;として削除するには、滅菌パスツールピペットを使用しています。 </li><li>蛍光細胞選別、必要に応じて、適切な細胞表面マーカー、さらに別の異なる細胞集団を用いて人口純度のフローサイトメトリーで細胞を分析します。必要に応じて分離した細胞は、さらなる分析のために今準備が整いました。 </li></ol> 3。代表的な結果三次元のスフェロイド共培養では、設計されたTiam1サイレンと線維芽細胞は、MAに増加した侵略を誘発する乳腺上皮細胞によるTRIX（ 図1、Dを比較して、E、FからA、B、C）。と線維芽細胞は、Tiam1の過剰発現は、乳癌細胞株SUM159（ 図1、G、H、IにJ、K、Lを比較して）減少して行列の浸潤を誘導する設計。 図2に示すように、一度共培養スフェロイドが確立されているが、細胞集団は、二次元の文脈で再めっきによる回転楕円体から単離することができる。テストセルの相対的な成長率に応じて、純粋な集団は、シリアル通路を通って出てくるかもしれません（ 図3に示すように）、または適切な細胞マーカーを使用してソートすることができます。共培養3Dから分離した後、上皮細胞は、分析のための十分な機会をできるように、長期間にわたって線維芽細胞との共培養により付与されたプロパティを保持します。この例では、Tiam1欠損線維芽細胞への暴露は、数週間持続した共培養HMECsの増加への移行を誘導される3D共培養（ 図4）からFTER分離。 <img alt="図1" src="/files/ftp_upload/3760/3760fig1.jpg" /> 図1光と蛍光顕微鏡下で共培養を確立回転楕円体のイメージング。スフェロイド培養は乳腺上皮細胞と線維芽細胞株を確立し、10〜14日間培養で成長する許可されています。 AF：上皮= HMEC-mCherry細胞。 GL：上皮細胞= SUM159-mCherry細胞。 AC、GI：線維芽細胞=コントロールRMF-GFP細胞。 DF：線維芽細胞= RMF-GFP Tiam1サイレンシングである。 JL：線維芽細胞= RMF-GFP Tiam1過剰発現した。矢印は、マトリックスに侵入上皮突起を示しています。 <img alt="図2" src="/files/ftp_upload/3760/3760fig2.jpg" /> 図2：マトリゲル培養からの分離後の光と蛍光顕微鏡下での文化のシリアルイメージング。 0日目はすぐに分離した後の画像を表します。1日目、25は、分離後の日を示しています。時間をかけて回転楕円体の増加を残して細胞のコロナ。最終的には非常に少数の細胞を用いた &quot;ゴースト&quot;回転楕円体（矢印）のまま。非常に少数のスフェロイドは、ゴーストを除去し、最初の通過した後に残っています。 <img alt="図3" src="/files/ftp_upload/3760/3760fig3.jpg" /> マトリゲル共培養からの分離後の細胞の図3フローサイトメトリー分析。スフェロイド共培養が成熟した後、細胞をピペッティングし、シリアル継代で3D共培養から分離されています。サンプルのアリコートをフローサイトメトリーによって分析されている（識別するために、緑色蛍光と赤色蛍光を使用して、この例では、GFPを発現するそれぞれの線維芽細胞とmCherryを発現する上皮細胞）が上皮細胞と線維芽細胞の相対的な集団を決定する。 <img alt="図4" src="/files/ftp_upload/3760/3760fig4.jpg" /> 図4。HMECsのトランスウェルの移行がisolati後も持続マトリゲル共培養から上に。純粋な集団を培養または蛍光細胞選別によって得られた後、必要に応じて、細胞を分析することができます。ここでHMECsいずれかのコントロール（C）または穿孔トランスウェル間のTiam1欠損（SHT）線維芽細胞と共培養の移行は、共培養からの分離後、2、4、8週目に評価した。

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利害の衝突が宣言されません。

ここで紹介する方法は、腫瘍細胞を含む上皮細胞に間質性線維芽細胞に及ぼす影響を分析する簡単な方法を表しています。さらに分析するためにマトリックスからの細胞の簡単な抽出を実現しながら、それは、細胞外基底膜マトリックスでサポートされている三次元のコンテキスト内で直接細胞間相互作用が可能になります。線維芽細胞の行が選択および上皮線のシグナル伝達経路に影響を与えるために操作することができるので、これは図1に示すように、侵襲性に変えることができ、腫瘍関連間質の研究に適用することができます。 3D共培養モデルは、細胞の侵襲性の変化が誘導されていることを視覚的に検証が可能になります。アイソレーション·プロトコルは、処理の最小の3D培養からの細胞の抽出を可能にし、セルのプロパティに影響を与える可能性トリプシンまたはマトリックス溶解剤の使用を避けることができます。 3D共培養における線維芽細胞への曝露の影響は後部上皮細胞に固執するその後の分析（ 図4）を容易に3次元共培養からERの分離、。ここに示す例では、細胞遊走への影響を示しながら、2Dで増殖する細胞に適用されるアッセイは、単離された細胞を使用することができます。我々は、関連付けられた乳腺上皮細胞の挙動に線維芽細胞Tiam1発現の変化の影響を分析するためにこのメソッドを使用しています。線維芽細胞におけるTiam1欠乏は、三次元培養および動物腫瘍モデル23の乳腺腫瘍細胞の浸潤や転移を促進します。このメソッドは、私たちは、分子の生化学的、機能的アッセイ（劉Buchsbaum、プレスで）を使用して、潜在的な関与する経路を分析することが可能になりました。 優れた健康で唯一の細胞株を使用し、プロセス中に文化の汚染を防止するために、厳密な無菌操作を遵守することが重要です。最良の結果が早いから細胞よりもむしろ遅れて通路を使用して取得されます（正確な通路数がspecifによって異なりますICは、セルライン）と50-80％コンフルエントの間に密度で採取した細胞から。マトリゲルの正確な処理がプロトコルに従って共培養を確立する前に両方を完全に液化を促進するために、細胞の添加前に行列の徹底した凝固を確保するために鍵となります。行列の不十分な量や固化が文化の中で組織化単分子膜としてではなく、回転楕円体で成長する細胞になります。第一共培養スフェロイドを確立するときに、井の中の最初の50μlのプラグインの配置が均等に分散スフェロイド形成への鍵です、最終的な行列のアンカー固体と、より均一な凝固を可能にします。自動化された細胞選別が混在する集団を分離するために使用する場合は、ポスト絶縁通路の手順ですべてのゴーストスフェロイドの除去が重要である。ここに示す例は、約10 mg / mlのタンパク質組成物とフェノールレッドフリーマトリゲルを使用しています。フェノールレッドフリーの準備は、ASSAの色を検出することができますこのような蛍光としてYS、。 このテクニックは、関連する上皮細胞の腫瘍または逆に関連した線維芽細胞上の腫瘍細胞に及ぼす影響に関する間質性線維芽細胞に及ぼす影響を研究するために細胞タイプの範囲に適用することができます。この手法は、複数の条件の下でin vitro細胞モデルでの迅速な評価を可能にし、対応する結果は、in vivoでの動物の組織モデルと同様にヒト組織サンプルで比較することができます。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> BDマトリゲル</td><td> BDバイオサイエンス</td><td> 356237 </td><td>フェノールレッドフリー</td></tr><tr><td>ハイクローン古典液体培地：DMEM F12 </td><td> ThermoScientific </td><td> SH30023.01 </td><td></td></tr><tr><td>ハイクローン子ウシ血清</td><td> ThermoScientific </td><td> SH3007303 </td><td></td></tr><tr><td>インスリン</td><td> EMDケミカルズ</td><td> 407709 </td><td> 0.005N HClに溶解し、0.22μmのフィルター</td></tr><tr><td>ヒドロコルチゾン</td><td> Sigma-Aldrich社</td><td> H4001 </td><td> 50％EtOHに溶解し、0.22μmのフィルターを</td></tr><tr><td> EGF </td><td> Sigma-Aldrich社</td><td> E9644 </td><td> 10mMの酢酸に溶解し、0.22μmのフィルターを</td></tr></tbody></table>

isolation of mammary epithelial cells from three-dimensional mixed-cell spheroid co-culture

膨大な努力が1-2正常および悪性細胞の行動に関与するシグナル伝達経路や分子を識別するに入っているが、この仕事の多くは、簡単に細胞操作を可能にする古典的な二次元の細胞培養モデルを用いて行われています。それは、細胞内シグナル伝達経路は、次元と3-4セルの表面張力を含む細胞外の力によって影響を受けていることが明らかになった。複数のアプローチがより正確に生物学的組織構造3を表している3次元モデルを開発するためにとられている。これらのモデルは、マルチ次元および建築ストレスを組み込む一方で、細胞への結果の効果の研究は、モデルの限界とその後の分析のために細胞を抽出することの難しさに起因する二次元組織培養で以下容易である。 腫瘍および腫瘍行動における腫瘍周囲微小環境の重要な役割Isはますます4を認識しつつ。腫瘍間質は、複数の種類の細胞と細胞外分子で構成されています。腫瘍の開発中に腫瘍細胞と間質細胞5の間の双方向の信号があります。腫瘍間質共進化に参加するいくつかの因子が同定されているが、体系的に識別し、これらの信号6の完全な配列を研究するための簡単なテクニックを開発する必要性が依然として存在する。線維芽細胞は、正常または腫瘍関連間質組織の中で最も豊富な細胞型であり、成膜と基底膜とパラクリン増殖因子7の維持に貢献しています。 多くのグループは、腫瘍治療への応答、免疫細胞の動員、シグナル伝達分子、増殖、アポトーシス、血管新生および浸潤8月15日を含む様々な細胞機能に線維芽細胞の役割を研究するために三次元培養システムを使用しています。私たちはお尻のために簡単な方法を最適化した混合細胞集団（共培養）16から22の三次元培養を作成するには、市販の細胞外マトリックスのモデルを使用して、乳腺上皮細胞上の乳腺線維芽細胞の影響をディエッシング。共培養を継続した細胞は、主にスフェロイドの外側に内部の線維芽細胞のクラスタリングおよび上皮細胞とスフェロイドを形成し、マトリックス中に多細胞の突起を形​​成する。変更された上皮細胞の浸潤につながる線維芽細胞の操作が容易に数値および上皮突起23の長さの変化によって定量することができる。さらに、我々は上皮挙動に及ぼす線維芽細胞への暴露の影響の分析を容易に三次元共培養上皮細胞を単離するための方法を考案した。我々は、複数のアッセイを実行するために十分な時間を許可し、共培養の効果は上皮細胞の分離後数週間持続することを発見した。この方法は、細胞への適応可能です悪性度を変化させず、特殊な装置を必要としません。この手法は、複数の条件の下でin vitro細胞モデルでの迅速な評価を可能にし、対応する結果がin vivoでの動物の組織モデルと同様にヒト組織サンプルで比較することができます。

Watch this Scientific Journal Video about Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture at JoVE.com

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

簡単な方法は、関連付けられている上皮細胞上の組織線維芽細胞に及ぼす影響を分析するために記述されています。このメソッドは、三次元組織培養の組み合わせにより、3Dから分離後の細胞の分析を容易にすることができます。技術は、腫瘍細胞上の腫瘍関連間質の効果の系統的な研究を可能にする、悪性度を変化させるセルに適用されます。

三次元混合細胞スフェロイド共培養から乳腺上皮細胞の分離

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

isolation-mammary-epithelial-cells-from-three-dimensional-mixed-cell

Nanyang Technological University

Research

JoVE Journal

Biology

16.0K ビュー.  Tufts Medical Center. 簡単な方法は、関連付けられている上皮細胞上の組織線維芽細胞に及ぼす影響を分析するために記述されています。このメソッドは、三次元組織培養の組み合わせにより、3Dから分離後の細胞の分析を容易にすることができます。技術は、腫瘍細胞上の腫瘍関連間質の効果の系統的な研究を可能にする、悪性度を変化させるセルに適用されます。

ビデオ: Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

マウス乳腺上皮細胞は乳腺刺激分化の過程でMammospheresを形成し、

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

生物学

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

マイクロ流体可溶性シグナルを介した相互作用を調査するために上皮細胞や細菌の共培養

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

乳腺上皮移植の手順

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

ヒト皮膚から大人上皮幹細胞の単離と培養

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA精製によってクロマチンの単離（チャープ）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

成体マウス切歯から歯科上皮幹細胞の単離および培養

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

三次元ハイドロゲルでイメージングをFRET

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

3次元共培養システムNICO-1を用いた卵巣癌幹細胞様細胞の血管遺伝学的特性の評価

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Ca2+過渡現象とL型カルシウム電流の同時測定のための高品質マウス心房および心室筋細胞の分離

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...