Name: an immunofluorescent method for characterization of barrett’s esophagus cells
Uploaded: 2014-07-20T15:00:00.000+00:00
Duration: 8 min 54 s
Description: Watch this Scientific Journal Video about An Immunofluorescent Method for Characterization of BarrettÃ¢â‚¬â„¢s Esophagus Cells at JoVE.com

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><tbody><tr><td>CP-A (Metaplastic Cell Line)</td><td>ATCC</td><td>CRL-4027</td><td></td></tr><tr><td>CP-B (High-grade Dysplastic Cell Line)</td><td>ATCC</td><td>CRL-4028</td><td></td></tr><tr><td>CP-C (High-grade Dysplastic Cell Line)</td><td>ATCC</td><td>CRL-4029</td><td></td></tr><tr><td>CP-D (High-grade Dysplastic Cell Line)</td><td>ATCC</td><td>CRL-4030</td><td></td></tr><tr><td>Keratinocyte-SFM (1x), Liquid</td><td>Life Technologies</td><td>17005-042</td><td></td></tr><tr><td>0.25% Trypsin-EDTA (1x), Phenol Red&amp;nbsp;</td><td>Life Technologies</td><td>25200056</td><td></td></tr><tr><td>Fetal Bovine Serum, Qualified, HI</td><td>Life Technologies</td><td>10438026</td><td></td></tr><tr><td>PSN antibiotic mixture</td><td>Life Technologies</td><td>15640</td><td></td></tr><tr><td>PBS - Phosphate-Buffered Saline</td><td>Life Technologies</td><td>10010049</td><td></td></tr><tr><td>Pro-long Gold Antifade Reagent with DAPI&amp;nbsp;</td><td>Life Technologies</td><td>P36931</td><td></td></tr><tr><td>Circular Glass Coverslip 18 mm</td><td>Fisher Scientific</td><td>15-183-86</td><td></td></tr><tr><td>&amp;beta;-catenin Primary Antibody (Rabbit)</td><td>Cell Signaling</td><td>9562S</td><td></td></tr><tr><td>Alexa Fluor 488 (Goat Anti-Rabbit)</td><td>Life Technologies</td><td>A11008</td><td></td></tr><tr><td>10 cm TC treated PS dish, sterile</td><td>USA Scientific</td><td>CC7682-3394</td><td></td></tr><tr><td>12-well TC treated PS plate, sterile</td><td>USA Scientific</td><td>5666-5180</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>276855</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma-Aldrich</td><td>A9434</td><td></td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>Bovine Serum Albumin - Fraction V</td><td>Sigma-Aldrich</td><td>85040C</td><td></td></tr><tr><td>SKI-606 (Bosutinib)</td><td>Selleck Chemicals</td><td>S1014</td><td></td></tr><tr><td>Square Bioassay Dish&amp;nbsp;</td><td>Thermo Scientific</td><td>240835</td><td></td></tr><tr><td>Parafilm&amp;nbsp;</td><td>VWR</td><td>82024-546</td><td></td></tr><tr><td>Disposable Pasteur Pipettes, Flint Glass</td><td>VWR</td><td>14672-380</td><td></td></tr><tr><td>Nexcelom Mini Cell Counter</td><td>Nexcelom</td><td></td><td></td></tr><tr><td>Cellometer Counting Chambers</td><td>Nexcelom</td><td>CHT4-SD100-014</td><td></td></tr><tr><td>Zeiss Apotome microscope&amp;nbsp;</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 BarrettÃ¢â‚¬â„¢s Esophagus Cells at JoVE.com

An Immunofluorescent Method for Characterization of Barrett&#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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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’s Esophagus Cells

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

ホルマリン固定パラフィン包埋ヒト卵黄扁平上皮癌試料におけるT細胞亜集団の四色蛍光免疫組織化学

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

がん研究

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification1.
A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs).
The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types2,3. However, no single &#x2018;marker&#x2019; has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. 
A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. 
In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response5, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody6. 
For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells7. The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.

We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray). Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes.

抗体マイクロアレイと蛍光多重を用いた大腸癌の細胞表面タンパク質のプロファイリング

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

免疫・感染学

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4&#8242;,6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.

A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology studies.

ホルマリン固定癌組織標本の腫瘍免疫研究の自動多重蛍光パネル

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of ...

Diagnosis of Neoplasia in Barrett&#8217;s Esophagus using Vital-dye Enhanced Fluorescence Imaging

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat and indistinguishable with white light imaging alone. As a result, a modality that highlights glandular architecture would be useful to discriminate neoplasia from benign epithelium in the distal esophagus. VFI is a novel technique that uses an exogenous topical fluorescent contrast agent to delineate high grade dysplasia and cancer from benign epithelium. Specifically, the fluorescent images provide spatial resolution of 50 to 100 &#956;m&#160;and a field of view up to 2.5 cm,&#160;allowing endoscopists to visualize glandular morphology. Upon excitation, classic Barrett&#8217;s metaplasia appears as continuous, evenly-spaced glands and an overall homogenous morphology; in contrast, neoplastic tissue appears crowded with complete obliteration of the glandular framework. Here we provide an overview of the instrumentation and enumerate the protocol of this new technique. While VFI affords a gastroenterologist with the glandular architecture of suspicious tissue, cellular dysplasia cannot be resolved with this modality. As such, one cannot morphologically distinguish Barrett&#8217;s metaplasia from BE with Low-Grade Dysplasia via this imaging modality. By trading off a decrease in resolution with a greater field of view, this imaging system can be used at the very least as a red-flag imaging device to target and biopsy suspicious lesions; yet, if the accuracy measures are promising, VFI may become the standard imaging technique for the diagnosis of neoplasia (defined as either high grade dysplasia or cancer) in the distal esophagus.

Vital-dye enhanced fluorescence imaging (VFI) is a novel in vivo technique that combines high-resolution epithelial imaging with exogenous topical fluorescent contrast to highlight glandular morphology and delineate neoplasia (high grade dysplasia and cancer) in the distal esophagus.

バイタル色素増強蛍光イメージングを使用して、バレット食道腫瘍の診断

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat ...

生物工学

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded&#160;tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image&#160;analysis technique.

パラフィン組織サンプルの単一細胞分析における免疫プロファイリングと空間マッピングの特性評価のための多重バーコード画像解析(英語)

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

人間の食道オルガノイドおよび気液界面の培養のプロトコール

Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and microbes. Maintaining the integrity of the epithelial barrier is critical for preventing infections and avoiding inflammation caused by harmless food-derived antigens. This article provides simplified protocols for generating human esophageal organoids and air-liquid interface cultures from patient biopsies to study the epithelial compartment of the esophagus in the context of tissue homeostasis and disease. These protocols have been significant scientific milestones in the last decade, describing three-dimensional organ-like structures from patient-derived primary cells, organoids, and air-liquid interface cultures. They offer the possibility to investigate the function of specific cytokines, growth factors, and signaling pathways in the esophageal epithelium within a three-dimensional framework while maintaining the phenotypic and genetic properties of the donor. Organoids provide information on tissue microarchitecture by assessing the transcriptome and proteome after cytokine stimulation. In contrast, air-liquid interface cultures allow the assessment of the epithelial barrier integrity through transepithelial resistance (TEER) or macromolecule flux measurements. Combining these organoids and air-liquid interface cultures is a powerful tool to advance research in impaired esophageal epithelial barrier conditions.

著者スポットライト:好酸球性食道炎の病態生理学の調査

Here, a protocol for the culture of human esophageal organoids and air-liquid interface culture is provided. Esophageal organoids' air-liquid interface culture can be used to study the impact of cytokines on the esophageal epithelial barrier.

好酸球性食道炎における上皮バリアを調べるための三次元細胞培養モデル 

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and ...

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, ...

生物学

Functional Characterization and Visualization of Esophageal Fibroblasts Using Organoid Co-Cultures

Epithelial stem and progenitor cells contribute to the formation and maintenance of the epithelial barrier throughout life. Most stem and progenitor cell populations are tucked away in anatomically distinct locations, enabling exclusive interactions with niche signals that maintain stemness. While the development of epithelial organoid cultures provides a powerful tool for understanding the role of stem and progenitor cells in homeostasis and disease, the interaction within the niche environment is largely absent, thereby hindering the identification of factors influencing stem cell behavior. Fibroblasts play a key role in directing epithelial stem and progenitor fate. Here, a comprehensive organoid-fibroblast co-culture protocol enabling the delineation of fibroblast subpopulations in esophageal progenitor cell renewal and differentiation is presented. In this protocol, a method to isolate both epithelial cells and fibroblasts in parallel from the esophagus is described. Distinct fluorescence-activated cell sorting strategies to isolate both the esophageal progenitor cells as well as the fibroblast subpopulations from either transgenic reporter or wild-type mice are outlined. This protocol provides a versatile approach that can be adapted to accommodate the isolation of specific fibroblast subpopulations. Establishing and passaging esophageal epithelial organoid mono-cultures is included in this protocol, enabling a direct comparison with the co-culture system. In addition, a 3D clearing approach allowing for detailed image analysis of epithelial-fibroblast interactions is described. Collectively, this protocol describes a comparative and relatively high-throughput method for identifying and understanding esophageal stem cell niche components in vitro.

Organoid-fibroblast co-cultures provide a model to study the in vivo stem cell niche. Here, a protocol for esophageal organoid-fibroblast co-cultures is described. Additionally, whole mount imaging is used to visualize the fibroblast-organoid interaction.

オルガノイド共培養を用いた食道線維芽細胞の機能特性評価と可視化

Epithelial stem and progenitor cells contribute to the formation and maintenance of the epithelial barrier throughout life. Most stem and progenitor cell ...

バレット食道の細胞の特徴のための免疫蛍光法

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ホルマリン固定パラフィン包埋ヒト卵黄扁平上皮癌試料におけるT細胞亜集団の四色蛍光免疫組織化学

抗体マイクロアレイと蛍光多重を用いた大腸癌の細胞表面タンパク質のプロファイリング

ホルマリン固定癌組織標本の腫瘍免疫研究の自動多重蛍光パネル

バイタル色素増強蛍光イメージングを使用して、バレット食道腫瘍の診断

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