この作品は、セントジョセフの財団（AJF、LJI）と米国肺協会、RG-224607-N（LJI）からの補助金によって支えられている。

1。細胞株のメンテナンス<ol><li>不死化ヒト転写酵素（TERT）タンパク質11ヒトテロメラーゼ逆の安定なトランスフェクションにより、高グレード異形成（CP-B、CP-C、CP-D）および化生（CP-A）細胞株BE。 </li><li>ウシ下垂体抽出物（BPE）、上皮成長因子（EGF）、5％ウシ胎児血清（FBS）、非必須アミノ酸（NEAA）、ペニシリン - ストレプトマイシン、ネオマイシン（PSN）、標準を補充したケラチノサイト培地中BE細胞株を維持する組織培養条件（37℃、5％CO 2、98％湿度）。 </li></ol> 2。細胞播種BE <ol><li> 12ウェルプレート/カバースリップの調製。ガラスシャーレやオートクレーブに18ミリメートルのカバースリップを配置。転写は、真空管に取り付けられた滅菌ガラスピペットを用いて12ウェル組織培養皿にの18mmカバーガラスオートクレーブ。滅菌PBSやメディアでカバーグラスを洗う。 </li><li>トリプシン処理し、不死化細胞を数える。細胞USIを数える自動細胞カウンターまたは血球計数器をngの。 20,000〜40,000細胞/ mlの濃度に培養培地中の細胞を希釈する。 </li><li>各ウェル20,000〜40,000個/ウェルの総細胞数のためのカバーガラスを含むへの細胞の1ミリリットルを配置します。 </li><li>優しく細胞がカバーグラスに付着することを保証するために井戸の底にカバースリップをプッシュする滅菌ピペットチップを使用してください。 </li></ol> BE細胞の3。薬物療法<ol><li>所望の濃度に暖かい（37℃）培地で選択された薬物を希釈し、繰り返し転倒やボルテックスミキサーを使用してよく混ぜる。経験的に決定された薬物濃度。カバーガラスの上の細胞の付着を可能にすることで、細胞の初期播種治療後24時間を開始</li><li>比較対照のために、培養培地中に希釈し、0.1％の車両（ジメチルスルホキシド[DMSO]または薬剤は、化合物を可溶化するために使用される）と一緒に細胞の追加のカバーガラスを扱う。薬剤を同定し、VEのための研究室のペンでラベルプレートhicle、サンプルを処理した。 37℃、5％CO 2、湿度98％に設定し、細胞培養インキュベーター中に細胞を置き、所望の時点インキュベートする。 </li></ol> 4。免疫蛍光染色<ol><li>固定<ol><li>薬物治療の所望のインキュベーション時間後、培地を吸引し、室温（RT、25℃）で1×リン酸緩衝生理食塩水（PBS）で迅速にカバースリップを洗浄する。 </li><li> PBSを吸引し、各ウェルに4％PFA [4％PFA / PBS]を含む1mlのPBSを添加し、室温で30分間インキュベートする。 </li><li> 4％PFA / PBSを吸引し、室温でPBSでカバーグラス3倍、5分間ずつ洗浄します。必要に応じて、4℃でのPBS/0.1％PFA中1週のための店のカバースリップ。 </li></ol></li><li>透過化/ブロッキング 抗体が細胞内標的にアクセスできるように固定されたPFAの細胞を透過。細胞外タンパク質を表面化するために、1×PBS/0.5％のBSA溶液に追加サポニンずに 、後続のすべての手順を実行します。 <ol><li>室温で10分間PBSで希釈した50のNH 4 Clでインキュベートすることにより、バックグラウンドを減少し、5分間に一度、RT PBSで洗う。 </li><li> 0.1％サポニンおよび0.5％ウシ血清アルブミン（BSA）を含むPBSの100〜300μlの30分間、細胞をインキュベートBE。水で調製し、4℃で保存し、濾過滅菌し、10％の在庫からサポニンを希釈</li></ol>注：これは、0.5％BSAの添加に起因するカバースリップを阻止することによって細胞タンパク質に対する抗体の非特異的結合を防止する。あるいは、抗体の非特異的結合を減らすために5％に希釈したヤギ血清をBSAに置き換えてください。 </li><li>人間の商工会議所の作製<ol><li>湿ったペーパータオルでチャンバーの底を積層して正方形のバイオアッセイディッシュを用意し、その上にパラフィルムの層を配置します。 </li><li>穏やか鉗子、シリンジ針を使用してパラフィルムカバースリップ上に、上向きに細胞を移す。 </li><li>カバースリップを識別するためのラボのペンでパラフィルムをマーク。 </li></ol></li><li>一次抗体インキュベーション<ol><li> 0.5％BSAおよび0.1％サポニンの両方を含有するPBS中で一次抗体を希釈する。 </li><li>優しく実験室の組織を使用してブロッキング溶液をオフにブロットおよび一次抗体溶液100μlを加える。 4℃で一晩BE細胞に一次抗体をインキュベート</li></ol></li><li>二次抗体インキュベーション<ol><li>スライドは室温で5分間ごとに、PBS/0.5％のBSA/0.1％のサポニンで3回洗浄します。 </li><li>二次抗体が一次抗体が発生された種を認識することをPBS/0.5％のBSA/0.1％のサポニン中の蛍光タグで結合させた二次抗体を希釈します。 </li><li>各カバーグラスに希釈した抗体を100μlを加える。室温で2時間、二次抗体をインキュベートする。 </li></ol></li><li>洗濯やカバーガラスの取り付け<ol><li> PBS/0.5％のBSA/0.1％のサポニンでカバースリップ3X、5分間ずつ洗浄します。洗浄緩衝液を除去し、カバーグラスをPBS 100〜300μlを加える。 注：二重反の場合体染色は、繰り返しての議論に概説された情報に応じて必要な追加の一次および二次抗体を用いて4.4から4.5を繰り返します。 </li><li>ピンセットを使用して、穏やかにカバーグラスを持ち上げ、実験組織またはペーパータオルを使用してPBSをオフブロット。 </li><li>ガラス顕微鏡スライド上に、細胞ダウン、カバーグラスに4を含む適切な退色防止封入剤 &#39;、6 - ジアミジノ-2 - フェニルインドール（DAPI）の15μLを加え、カバースリップを反転。 </li><li>スライドフォルダにスライドを置き、ハードセット退色防止媒体が硬化させるために室温で一晩直射光からインキュベート、メーカーの指示に従って。抗退色媒体がセットされた後、スライドを顕微鏡的視覚化するまで4℃又は-20℃のいずれかで保存することができる。 </li></ol></li></ol>カバーガラスの5。顕微鏡可視化染色された細胞を、共焦点顕微鏡またはIFが可能なアップライトmicroscopのいずれかを介して画像化することができるE。共焦点顕微鏡は、離散深度で高解像度の画像を提供するが、直立顕微鏡が速く有用な画像を生成することができる。簡潔にするために、イメージングの方法は、標準的な顕微鏡を提示されている使用して細胞であっても染色した。一般的には、画像をフルオレセインイソチオシアネート（FITC）または類似のフルオロフォア（ すなわち緑色蛍光タンパク質）に、テキサスレッドおよびDAPIは、これらのフルオロフォアを識別することのできるフィルタを備えた顕微鏡を必要とします。互換性の蛍光プローブを決定するためにプロトコルを開始する前に、顕微鏡をチェックしてください。 <ol><li>標準IF顕微鏡イメージング<ol><li> -20℃または4℃から保存されたスライド/カバースリップを削除し、それらを室温に加温することができます。顕微鏡と水銀アークランプの電源をオンにします。 </li><li>カバーガラスは、顕微鏡ステージ上に目的の方に向けた状態で顕微鏡スライドを配置します。油浸対物レンズを使用するために、カバーガラス上にイマージョンオイルの滴を追加します。 </li><li> DAPIフィルターを選択し、シャッターを開いてください。 </li&gt; <li>画像が表示されるまで、接眼レンズを通して見ながら目的の焦点を合わせる。すべての細胞はDAPIで染色されるので、核は容易に観察する必要があります。 </li><li>各画像処理ソフトウェアを用いて画像を収集する。 </li></ol></li></ol>

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著者は、彼らが競合する経済的利益を持っていないことを宣言します。

我々は、これらの細胞に対する標的療法の生理学的効果の解明に向けたBE細胞のIFを適用するための方法を説明してきました。我々はBE細胞に対してこれらの手順の使用を記載してきたが、我々は、これらの方法はまた、異なる細胞型13,17,18様々に適用可能であることを見出した。また、これらの手順は、特定のターゲットのための染色を最適化するいくつかの方法で変更することがで...

バレット食道（BE）は、食道の正常扁平上皮および胃食道逆流症（GERD）1から生じる胃内容への慢性暴露の結果での化生変化です。 BEは、GERDへの反応に対する防御機構であると考えられているがBEの存在は、食道腺癌（EAC）、有意に低い生存率1を搬送する疾患のリスクの増加を与える。現在の推定は、米国人口の5.6％がBEていることを示唆しているBEは、しばしば無症候性であるように、しかしながら、それはBEの大部分は2診断未確定のままであると考えられる。逆流性食道炎とEACの両方の発生率は、継続的な成長3を見てきたように、この情報が潜在的にEACにBEの進行を防止に向けた治療的アプローチを提供することが特に、EACにBEの進行に関与する分子メカニズムを理解することが重要になってきている。 1の現在のパラダイムをもたらした。慢性炎症、外傷、および胃内容物への長期暴露に起因する遺伝毒性損傷はEACに腫瘍の進行を促進BE病変に対する選択圧を及ぼす。遺伝子変異の数は、EAC進行することで中に同定されている。しかし、これにもかかわらず、細胞のシグナル伝達における正確な変化および細胞構造および機能に対するその後の影響についての明確な理解の欠如がある。 インビトロ細胞株において不死化に特に標的化治療化合物の効果を調査において、細胞シグナル伝達の効果を研究するための有用なツールである。 BE不死細胞株の最近の商業的利用可能性は、そのような研究を可能にする。そのような広く使用されている生存率アッセイなどのハイスループットアッセイは、細胞増殖およびsuの際に標的療法の効果を評価する上で貴重であることができるが4-6 rvival、これらのアッセイは、細胞の形態に依存細胞シグナル伝達の効果を問い合わせるために有用ではない。免疫蛍光染色（IF）は、細胞の形態、増殖、および生存特性および関与するタンパク質の際に標的療法の効果を調査するための有用な技術である。当研究室では、薬物治療7の可能な化学予防治療とバイオマーカーの輪郭を描くことを望んでも細胞株の際に臨床的に利用可能な薬剤の効果を評価するためにIFを用いて、不死化細胞株に向けて、これらの方法を適応している。同様に、EACでのこれらの技術の適用は、BEと食道不死化細胞株は、EAC進行8-10にBEに関する重要な知見を描写している。標的療法で処置したBE細胞の分析は、細胞構造およびタンパク質局在化の変化の特徴付けを可能にする、値を有するIF我々は見つける。ここでは、Cに不死化細胞のIFのための私達の方法を提示薬物治療をharacterize。

<table border="0" cellpadding="0" cellspacing="0" width="743">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Name of the Material/Equipment</td>
			<td style="width:116px;">Company</td>
			<td style="width:108px;">Catalog Number</td>
			<td style="width:224px;">Comments/ Description (optional)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CP-A (Metaplastic Cell Line)</td>
			<td>ATCC</td>
			<td>CRL-4027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CP-B (High-grade Dysplastic Cell Line)</td>
			<td>ATCC</td>
			<td>CRL-4028</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CP-C (High-grade Dysplastic Cell Line)</td>
			<td>ATCC</td>
			<td>CRL-4029</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CP-D (High-grade Dysplastic Cell Line)</td>
			<td>ATCC</td>
			<td>CRL-4030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Keratinocyte-SFM (1X), Liquid</td>
			<td>Life Technologies</td>
			<td>17005-042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.25% Trypsin-EDTA (1X), Phenol Red&#160;</td>
			<td>Life Technologies</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum, Qualified, HI</td>
			<td>Life Technologies</td>
			<td>10438026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PSN ANTIBIOTIC MIXTURE</td>
			<td>Life Technologies</td>
			<td>15640</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS - Phosphate-Buffered Saline</td>
			<td>Life Technologies</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pro-long Gold Antifade Reagent with DAPI&#160;</td>
			<td>Life Technologies</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Circular Glass Coverslip 18mm</td>
			<td>Fisher Scientific</td>
			<td>15-183-86</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-catenin Primary Antibody (Rabbit)</td>
			<td>Cell Signaling</td>
			<td>9562S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexafluor 488 (Goat Anti-Rabbit)</td>
			<td>Life Technologies</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10cm TC treated PS dish, sterile</td>
			<td>USA Scientific</td>
			<td>CC7682-3394</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">12-well TC treated PS plate, sterile</td>
			<td>USA Scientific</td>
			<td>5666-5180</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Saponin</td>
			<td>Sigma-Aldrich</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumin - Fraction V</td>
			<td>Sigma-Aldrich</td>
			<td>85040C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SKI-606 (Bosutinib)</td>
			<td>Selleck Chemicals</td>
			<td>S1014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Square Bioassay Dish&#160;</td>
			<td>Thermo Scientific</td>
			<td>240835</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Parafilm&#160;</td>
			<td>VWR</td>
			<td>82024-546</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Disposable Pasteur Pipets, Flint Glass</td>
			<td>VWR</td>
			<td>14672-380</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nexcelom Mini Cell Counter</td>
			<td>Nexcelom</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cellometer Counting Chambers</td>
			<td>Nexcelom</td>
			<td>CHT4-SD100-014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zeiss Apotome microscope&#160;</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an immunofluorescent method for characterization of barrett&#8217;s esophagus cells

食道腺癌（EAC）は、全生存期間の17％未満の割合とEACの発生率は過去20年間に劇的に上昇しているがあります。 EACの主要な危険因子の一つは、バレット食道（BE）、慢性の胸焼けに応答して正常食道扁平上皮化生の変化である。 EACとBE、EAC者になるの進行に関与する分子事象、特に変更されたシグナル伝達経路の尋問の間に十分に確立された接続にもかかわらず、ほとんどわかっていない。これの多くは、これらの疾患を研究するために利用できるin vitroモデルにおいて適切性の欠如に起因している。最近では、不死化細胞株BEは、BEのインビトロ研究を可能にする商業的に利用可能になっている。ここでは、治療用化合物への曝露後の細胞シグナル伝達および構造体のインビトロ特徴付けを可能に 、BE不死化細胞株の免疫蛍光染色のための方法を提示する。これらの技術の適用は、HELうpはEACの進行者になるに関与するメカニズムへの洞察を開発し、EACの治療および予防のための潜在的な道を提供する。

記載された手順の適用から得られた結果の例を、 図1A〜Dに示されている。 CP-DおよびCP-Cを用いたカバーガラスの細胞は24 Srcファミリー阻害剤の1μM、SKI-606との時間、またはビヒクル（DMSO）処理し、ヘレン·ジャンクショ​​ンおよびWntシグナル伝達タンパク質、βについて上記の手順を用いて染色したBE -カテニン。細胞核の標識はDAPI（青色）で標識することによって達成しなが?...

Watch this Scientific Journal Video about An Immunofluorescent Method for Characterization of Barrettâ€™s Esophagus Cells at JoVE.com

An Immunofluorescent Method for Characterization of Barrett&amp;#8217;s Esophagus Cells

バレット食道の分子ドライバーを改良し、情報のための識別可能な必要性がある。免疫蛍光染色は、細胞形態学上の細胞シグナル伝達の効果を理解するために有用な技術である。私たちは、バレット食道細胞において治療上の処置を評価するための免疫蛍光染色を使用するためのシンプルで効果的なプロトコルを提示する。

バレット食道の細胞の特徴のための免疫蛍光法

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One ...

an-immunofluorescent-method-for-characterization-barretts-esophagus

Research

JoVE Journal

Biology

An Immunofluorescent Method for Characterization of Barrett&#8217;s Esophagus Cells

14.0K ビュー.  St. Joseph's Hospital and Medical Center. バレット食道の分子ドライバーを改良し、情報のための識別可能な必要性がある。免疫蛍光染色は、細胞形態学上の細胞シグナル伝達の効果を理解するために有用な技術である。私たちは、バレット食道細胞において治療上の処置を評価するための免疫蛍光染色を使用するためのシンプルで効果的なプロトコルを提示する。 

ビデオ: An Immunofluorescent Method for Characterization of Barrett&#8217;s Esophagus Cells

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

昆虫細胞における組換えmCD1dタンパク質の製造方法。

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

生物学

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

培養胚のための方法<em&gt; C.虫</em&gt;細胞

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

エキソソームの定量化とサイズの測定のための革新的方法

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

マウスおよびヒト食道から筋線維芽細胞の単離

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

免疫組織化学および免疫蛍光分析のための腸組織の調製のための改良されたスイス・ローリングテクニック

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

工場ポリソームプロファイリングのための簡単​​な方法

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

ゼブラフィッシュにおける腎毒素マイクロインジェクションは急性腎障害をモデルに

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

動的細胞突起におけるソフトウェア支援トラッキングのためのグラフィカルユーザインタフェース

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

筋幹細胞におけるオートファジーのin situ Immunofluorescent Staining

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.