This work was supported by an NSF/ARRA award (IOS 0920357) and an award from the NIH (5R21MH098463).

1。細胞調製（日-2〜0） <ol><li>分割HEK293細胞1： - 80％コンフルエント（2 - 3日間）および0.05％トリプシン-EDTA溶液を用いて5〜60まで、5％CO 2、37℃で増殖培地中でインキュベートする。各条件については、2×100mm皿内の培養細胞。 </li></ol> 2細胞トランスフェクション（1日目） <ol><li>そのようなリポフェクタミンなどのトランスフェクション試薬を使用してコードするプラスミドFc融合でHEK293細胞をトランスフェクトする。同等のトランスフェクション効率をもたらす代替的な方法を用いてもよい。 </li><li> 24時間インキュベーターにトランスフェクトされた細胞を返します。 </li></ol> 3セルの伝播（2日目） <ol><li> 37℃までのウシ胎児血清（-FBS）なしの予熱増殖培地。 </li><li>料理は10ミリリットルの成長メディア-FBSで2回すすぎ、1時間、37℃のインキュベーターに皿を返す。 </li><li>合計3回の洗浄のために、10ミリリットルの成長メディア-FBSと文化の料理をもう一度洗浄します。 </li><li> Incuお譲りしたメディアを収集する前に、FBSを含まない別の48時間、37℃でトランスフェクトされたHEK293細胞。 </li></ol> 4。ビーズ集約（4日目） <ol><li>培養皿からメディアを収集するには、細胞破片をペレット化し、5分間500×gで50ミリリットルコニカルチューブとスピンに料理の各ペアからメディアを転送します。 </li><li> 30ミリリットルの注射器、0.45μmのシリンジフィルターを用いて遠心フィルターに50ミリリットルコニカルチューブからフィルターメディア。 </li><li>濃縮された培養培地の体積は約500μlの（約15分）まで、4,000×gで、4℃で遠心分離フィルタースピン。すべての培養培地を添加して、濃縮されるまで繰り返します。 </li><li> 、各サンプルについて1.5mlのマイクロチューブに結合バッファーの氷冷1mlにプロテインG磁気ビーズを1.5μl加え、磁石上の場所とバッファを削除します。すぐにプロテインG磁気ビーズに濃縮した培養培地を追加します。 </li><li> 2時間、4℃で管を回転させます。 </li><li> Plの磁石上のエースチューブとは、メディアを取り出します。すばやく結合バッファーの氷冷1で2回ビーズを洗浄した後、結合緩衝300μlのビーズを懸濁します。 </li><li>スプリットは2管、各チューブに150μlの中にビーズを再懸濁し、その後、それぞれ、「カルシウム」と「いいえカルシウム」の条件について200のCaCl 2または200のEDTA、1.5μlを添加する。 </li><li>うつ病への転送、各条件からの100μlをうまくスライドさせ、透過光顕微鏡（ 図2）を使用して、顕微鏡写真を収集します。所望の各時点での各実験のために5視野からの画像を収集します。 </li></ol> 5。データ解析<ol><li> [ファイル ]プルダウンメニューから... ImageJの、または同等の画像解析ソフト、 インポート/イメージシーケンスを使用して、5つの画像データセットのオープン1を使用した。これらは、画像スタックとして開かれます。 </li><li> イメージ / プロパティ...]ダイアログボックスで、私を変更ピクセルにメイジユニットおよび1.0にピクセルの幅と高さをピクセル単位で設定します。 </li><li> 画像プルダウンメニューで調整/しきい値...コマンドを使ってバイナリに画像を変換します。ビーズやビーズ凝集体に貢献する画素が含まれるようにしきい値を設定しますが、その背景と小さい粒子を除外します。スタック内のすべての画像に適用します。 </li><li> セットの測定 ]ダイアログボックスで、 エリアとスタックポジションチェックボックスをオンにします。 </li><li>プルダウンメニューの分析に...粒子を分析し実行します。これは、それらの大きさ（面積）およびそれらが同定された画像を含む同定された粒子のリストを生成する。 </li><li>実験と実験条件について、この手順を繰り返します。 </li></ol>

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The authors declare that they have no competing financial interests.

細胞接着は多細胞生命の本質的な特徴であり、細胞表面タンパク質の広範な配列によって媒介される。このうち、詳細な接着性が比較的小さい割合と理解される。ここでは、便利なエピトープタグに融合された分泌された外部ドメインの同種親和性接着剤能力を調査するための、シンプルで迅速なプロトコルを記載している。このアプローチは、多くの重要な利点を有している。タンパク質?...

細胞間接着は、多細胞生物の発生および完全性に必須であり、細胞表面分子の多様な配列によって媒介される。これらの接着分子の多くは、多くはまだ発見されていないものの、同定され、特徴付けられている。いくつかの方法は、アッセイを、細胞選別、細胞凝集アッセイ1-8と、原子間力顕微鏡、表面力分光法9-12のような生物物理学的方法を含む細胞接着分子（CAM）の特性を調べるために使用される。 偶数細胞株を用いてインビトロ系において簡略化の複雑さは、それが困難な推定上のCAMの接着特性を決定することを可能にする。非接着細胞株にトランスフェクトした場合には細胞凝集を誘導する場合、通常、分子はCAMと考えられている。しかし、これは接着活性の直接的な証拠はないことは明らかである。例えば、細胞表面の配信または安定性を促進するCAMはまた、増加した細胞凝集1,13をもたらすであろう。また、真のCAMは、細胞株は、細胞表面送達または安定化に必要なその他の補助因子を欠いている場合、細胞凝集を媒介することに失敗することがあります。 これらの複雑な要因を回避するために、より直接的なアッセイは、接着性相互作用は、細胞外ドメインの固有の生化学的性質であるべきであるという考えに基づいていることを採用することができる。ビーズは最初に呉-CAM 2を特徴付けるために使用されたが、これらのアッセイは、カドヘリン介在性接着12,14,15を調べるために拡張されている。 C-カドヘリン細胞外ドメイン-Fc融合の融合を使用して、Gumbinerラボは、複数のカドヘリンリピートが相互作用14を同種親和性に寄与することを示した。 ショウジョウバエからプロト1,15-18とDSCAMアイソフォームの数を持っているように、同等のビーズ凝集アッセイ、E-カドヘリンとN-カドヘリン接着を使用すると、、12,15を特徴づけられている<em&gt; 19。ここでは、推定上の同種親和性のCAM（ 図1）の分泌され、エピトープタグの外部ドメインの接着活性を特徴付けるため、比較的簡単かつ迅速なアッセイを記述する。私たちは、カドヘリンスーパ​​ーファミリーのメンバーを特徴づけるために、主にこのアッセイを使用している。

<table><tbody><tr><td>pFc-N1 plasmid</td><td>Addgene</td><td>&lt;span style=&quot;color:red;&quot;&gt;&lt;strong&gt;To be submitted&lt;/strong&gt;&lt;/span&gt;</td><td></td></tr><tr><td>HEK293 cells</td><td>American Type Culture Collection</td><td>CRL-1573</td><td></td></tr><tr><td>DMEM</td><td>Mediatech - Corning</td><td>10-013-CV</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma</td><td>F2442</td><td></td></tr><tr><td>100x Pen-Strep (10,000 U/ml)</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Life Technologies</td><td>25300054</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Life Technologies</td><td>11668030</td><td></td></tr><tr><td>Dynabeads &amp;ndash; Protein G</td><td>Life Technologies</td><td>10003D</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A3294</td><td></td></tr><tr><td>Depression slides</td><td>Electron Microscopy Sciences</td><td>71878-06</td><td></td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-10 membrane</td><td>Millipore</td><td>UFC901024</td><td></td></tr><tr><td>0.45 mm syringe filter</td><td>Sarstedt</td><td>83.1826</td><td></td></tr><tr><td>Dynamag-2 Magnet</td><td>Life Technologies</td><td>12321D</td><td></td></tr><tr><td>Fiji (ImageJ) Image Analysis Software</td><td>&lt;a href=&quot;http://fiji.sc/Fiji&quot;&gt;http://fiji.sc/Fiji&lt;/a&gt;</td><td></td><td></td></tr><tr><td colspan="4">Table of Buffers/Solutions</td></tr><tr><td>Growth Media</td><td>DMEM + 10% FBS + Pen-Strep</td><td>Filter sterilize and store at 4 &amp;deg;C.</td><td></td></tr><tr><td>Growth Media &amp;ndash;FBS</td><td>DMEM + Pen-Strep</td><td>Filter sterilize and store at 4 &amp;deg;C.</td><td></td></tr><tr><td>Binding Buffer</td><td>50 mM Tris, pH 7.4, 100 mM NaCl, 10 mM KCl, 0.2% BSA</td><td>Vortex until dissolved, keep on ice.</td><td></td></tr></tbody></table>

bead aggregation assays for the characterization of putative cell adhesion molecules

細胞 - 細胞接着は多細胞生命の基本であり、細胞表面タンパク質の多様な配列によって媒介される。しかし、これらのタンパク質の多くのための接着剤の相互作用はほとんど解明されていない。ここでは、推定される同種親和性細胞接着分子の接着特性を特徴付けるための、シンプルで迅速な方法を提示する。培養されたHEK293細胞は、細胞表面タンパク質の分泌、エピトープタグ外部ドメインをコードするDNAプラスミドでトランスフェクトされる。エピトープタグに特異的な官能化されたビーズを用いて、可溶性の分泌された融合タンパク質を培地から捕捉される。コー​​ティングされたビーズは、その後、ビーズ凝集アッセイにおいて、または同種親和性接着を試験するためのアッセイを選別蛍光ビーズに直接使用することができる。所望の場合、突然変異誘発は、その後の接着に必要な特定のアミノ酸又はドメインを解明するために使用することができる。このアッセイは、発現されたタンパク質の少量のみを必要とする安定な細胞株の産生を必要とせず、アコすることができ4日間でmplished。

<html>実験例は、FC（NcadEC-Fc）のに融合したN-カドヘリンの細胞外ドメインによってカルシウム依存性ビーズの凝集を示した図2に示されている。カルシウムの非存在下では、ビーズは凝集するほとんど又は全く傾向を示す、時間（ 図1A、C）との凝集体サイズの増加はない。カルシウムの存在下では、ビーズは、凝集体サイズが（ 図1B、C）は、時間の経過と...

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Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

推定細胞接着分子のキャラクタリゼーションのためのビーズ凝集アッセイ

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

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10.5K ビュー.  Ohio State University. Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

ビデオ: Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ability to accurately classify and quantify cell extensions and measure the effect on a cell&#39;s adhesive capacity is critical to determining how cell-signaling events impact cancer cell behavior and aggressiveness. Here, we describe the in vitro design and use of a cell extension quantification method in conjunction with an adhesion capacity assay in an established in vitro model for adrenocortical carcinoma (ACC). Specifically, we test the effects of DKK3, a putative tumor suppressor and a pro-differentiation factor, on the membrane extension phenotype of the ACC cell line, SW-13. We propose these assays to provide relatively simple, reliable, and easily interpretable metrics to measures these characteristics under various experimental conditions.

This study demonstrates the utility and ease of quantitative cell membrane extension measurement and its correlation to adhesive capacity of cells. As a representative example, we show here that Dickkopf-related protein 3 (DKK3) promotes increased lobopodia formation and cell adhesiveness in adrenocortical carcinoma cells in vitro.

細胞膜の拡張機能と癌細胞の接着の役割を勉強のキャラクタリゼーション

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ...

がん研究

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

用接着周波数アッセイその場で</em細胞間のインタフェースにおけるクロス接合分子間相互作用の>速度論解析

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

生物工学

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

細菌 - 宿主相互作用を特徴づけるために、バイオミメティック材料

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

免疫・感染学

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Tリンパ球接着分子の相互作用の研究のための接着性の下でせん断応力の測定

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

炎症性腸疾患における癒着防止治療の機能分析のための動的接着アッセイ

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse formation and maintenance. Many of these fundamental interactions occur in trans and are typically induced by heterophilic or homophilic interactions between cells expressing membrane anchored binding pairs. Elucidating how disease relevant mutations disrupt these fundamental protein interactions can provide insight into a myriad of cell biology fields. Many protein-protein interaction assays do not typically disambiguate between cis and trans interactions, which potentially leads to an overestimation of the extent of binding that is occurring in vivo and involve labor intensive purification of protein and/or specialized monitoring equipment. Here, we present an optimized simple protocol that allows for the observation and quantification of only trans interactions without the need for lengthy protein purifications or specialized equipment. The HEK cell aggregation assay involves the mixing of two independent populations of HEK cells, each expressing membrane-bound cognate ligands. After a short incubation period, samples are imaged and the resulting aggregates are quantified.

Here, we present an optimized protocol to rapidly and semiquantitatively measure ligand-receptor interactions in trans in a heterologous cell system using fluorescence microscopy.

異種細胞系におけるタンパク質凝集による細胞間相互作用の測定

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse ...

神経科学

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮細胞接着性の定量<em&gt;インビトロ</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

生物学

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

磁気ビーズを使用した機械的張力に応答して細胞表面接着リフォームの分析

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to the substrate as a function of the number and size of focal adhesions, and cell shape, characterized by the cell shape index and other shape descriptors. Focal adhesions were visualized by paxillin staining and cell-cell borders were marked by junction plakoglobin and actin. The methods for cell culture and staining were standard; images represent single focal planes; image analysis was performed using publicly available image processing software. The presented protocols are used to quantify the number and size of focal adhesions and the differences in cell shape distribution in the monolayers, but they can be repurposed for the quantification of the size and shape of any other distinct cellular structure that can be stained (e.g., mitochondria or nuclei). Assessing these parameters is important in the characterization of the dynamic forces in adherent cell layer, including cell adhesion and actomyosin contractility that affects cell shape.

The article describes quantification of 1) the size and number of focal adhesions and 2) cell shape index and its distribution from confocal images of the confluent monolayers of MCF7 cells.

MCF7細胞単層における細胞基質接着面積と細胞形状分布の定量化

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

推定細胞接着分子のキャラクタリゼーションのためのビーズ凝集アッセイ

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