著者は、読書と原稿のコメントを Bhagawat サブラマニアン、李 (ジェイソン) チャン ポール ランダッツォとキャロル ・親に感謝したいです。この作品は、国立がん研究所の学内研究プログラムによって支えられた健康の国民の協会。

1. 料理 (1 日目) のコーティング
注: しないように取り扱いにプレートの底に指紋や汚れを残します。
<ol><li>マウスのコラーゲン氷の上の IV を解凍し、それを希釈 50 mM HCl (pH 1.3) 10 μ g/mL コラーゲン IV 溶液 3 mL を準備します。 注: コラーゲンを 37 ° C で沈殿させるしたがって、コラーゲン溶液を低温融解と使用中保つことが重要です。適切なサイズの因数を準備することによって凍結融解の繰り返しを避けるため-80 ° C で保存して</li>
	<li>12 ウェル ガラス底板の各ウェルに 250 μ L コラーゲン IV ソリューションを追加し、適切なサイズ、タイトな終了のプラスチック ボックスにそれらを置きます。湿らせたペーパー タオル上と湿気のある商工会議所を製造し、ボックスを プレートの周りに配置します。室温で一晩版を孵化させなさい。 注: 成長分野だけは塗装する必要がある、それはコラーゲン溶液と、プレートの下部に取り付けられているカバー スリップをカバーするのに十分な (図 1A、Bのために板の回路図を参照してください)。</li>
	<li>次の朝は、脱イオン H2O 2 回非吸収コラーゲンとバッファーを削除するとプレートをすすいでください。まあ、プレート下部に配置され、coverslip によって形成される (場合、水はそれがスプラッシュはこの領域に戻) 端にプレートの端に水を指示します。層流フードに板を風乾します。すぐに板を使用したり、4 ° C で最大 5 日間保存します。 注: 異なる細胞は異なるコーティング、フィブロネクチン、コラーゲンなど私を必要があります。</li>
</ol>2. めっきドット アッセイ (2 日目)
<ol><li>
細胞懸濁液を準備します。
	<ol>
<li>細胞懸濁液を準備するには、5% 80% 合流馬血清を添加した DMEM/F12 中 60 mm 細胞培養用ディッシュで栽培されているセルを使用します。</li>
		<li>カルシウムとマグネシウム無料ダルベッコ リン酸緩衝生理食塩水 (DPBS) で細胞を濯ぎ 1 回 500 μ L トリプシン-EDTA (常温) を追加し、37 ° C、5% CO2、加湿雰囲気でインキュベーターで 3-5 分のセルを孵化させなさい。</li>
		<li>トリプシン処理を停止し、カウントを 20 4.5 mL 培養液中の剥離細胞を中断、検定に細胞懸濁液の μ 因数。</li>
		<li>3 分間 200 × g で遠心分離によっての 15 mL の円錐管の残りのセルのペレット、上清を吸引、3 x 106セル/mL に培養液中の細胞を再懸濁します。 注: その他の基材をコーティングや細胞の最適な細胞濃度は希釈系列の確立されなければなりません。3 x 106セル/mL は、出発点として推奨されています。</li>
	</ol>
</li>
	<li>
めっきセル
	<ol>
<li>コラーゲン IV の各カバー スリップの中心に細胞懸濁液の 10 μ L のドロップの場所 (図 1A, B) の塗装を傷に触れたりなし 12 ウェル ガラス底板にコーティング。プレートを付けるセルを許可するように、加湿雰囲気 37 ° C で 30 分間インキュベートします。倒立顕微鏡で細胞が接続されているを確認します。 注: 最高といえる、ドロップの端、単分子膜の形成は、(図 1C) を観察することができます。必要な場合は、プレートを孵化させなさいまで 3 h するインキュベーターの湿度が十分に高く、滴がすぐに乾燥してください。このステップの間にドロップ量の減少が見られる場合は、湿度を高める井戸間のスペースに PBS を追加します。</li>
		<li>非添付のセルを削除する 2 回培養液 1 mL と井戸を優しく洗います。各ウェルには、培地 1 mL を追加します。ない浮遊細胞が残る、そうでなければもう一度洗浄を倒立顕微鏡で確認します。一晩で、37 ° C、5% CO2、加湿雰囲気、明確に定義された細胞シートを取得するセルを孵化させなさい。 注: 浮遊細胞はプレートに再アタッチして不要な細胞群れ体を形成する可能性があります。</li>
	</ol>
</li>
</ol>3. 刺激細胞 (3 日目)
<ol><li>逆顕微鏡を使用してコロニーが正しく成長しているセルを確認するすべての井戸を確認します。必要に応じて、不適切な井戸をマークし、それらを使用しないでください。</li>
	<li>調査条件ごとに適切なプレートの各ウェルにラベルを付けます。重複した、または 3 通で各条件を実行します。</li>
	<li>0.1% 馬血清 (飢えた中) を含む DMEM/f12 キーでセルを餓死に媒体を変更 3 h 前に細胞が刺激されます。</li>
	<li>EGF (最終濃度が 5 ng/mL) の添加、または他の目的の仲介によって細胞を刺激し、優しく 1 mL micropipettor を使用してメディアをミックス、プレートを旋回します。</li>
	<li>セクション 4.1 で説明した刃先変位とのエッジの輪郭を観察する 1-4 日のプレートを孵化させなさいまたはセクション 4.2 で説明されているようにタイムラプス イメージングを実行します。</li>
</ol>4. 細胞群れ体と細胞移動の可視化
<ol><li>
ヘマトキシリン ・ エオジン (HE) 染色コロニー成長 (4-7 日目) を測定するには
	<ol>
<li>70% エタノール (1 mL/ウェル) ~ 2 分のセルを修正します。</li>
		<li>ヘマトキシリン (1 mL/井戸)、~ 2 分で核を染色します。</li>
		<li>洗浄細胞核まで水道水 (1 mL/も) が青、5-10 分。</li>
		<li>エオシン (1 mL/井戸)、~ 2 分で細胞質を染色します。</li>
		<li>(1 mL/井戸) の脱イオン水ですすいでください。 注: ヘマトキシリンとエオシンのソリューションすることができます収集し、かすかな染色になるまで複数回使用します。</li>
		<li>プレートを風乾します。</li>
		<li>プレートを反転し、ドットの直径を定規で測定します。また、カメラ プレートの写真を撮るし、ImageJ でドットの直径を分析します。</li>
	</ol>
</li>
	<li>
微速度顕微鏡観察 (日 3)
	<ol>
<li>インキュベータ顕微鏡に切り替えるし、37 ° c. に温度を調整DH25 %o. 調整 CO2で加湿器を満たしなさい。インキュベーターのチャンバーを加湿して電動ステージを自由に移動できることを確認します。インキュベーターのチャンバーを閉じるでき、37 ° c. に調整する顕微鏡 注: 顕微鏡を使用 (材料表参照) ここで、セルがフォーカスの漂流を避けるためにイメージを作成する前に、この少なくとも 3 h の 37 ° C まで加熱する必要があります。</li>
		<li>インキュベータ顕微鏡のステージにプレートを置き、ステージ リストを設定します。画像 2 つの反対端と各セル ドット (図 1A) の中心。</li>
		<li>対物レンズ 10 倍を使用して 15-24 h の 3 分毎の画像を取る。データをダウンロードして、選択したプログラムで画像解析を続行などImageJ や Matlab。</li>
	</ol>
</li>
</ol>

<ol>
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著者は、何を開示します。

腫瘍の進行の間に周囲の組織に侵入し、15遠隔部位に転移する起源の組織から移行します。細胞の細胞群からの移動は、ドットのアッセイで観察できます。ここでは、ドットの試金は EGF に応えてひと乳癌細胞の渡り鳥の表現型の分析によって例証されます。
ドットのセットアップでいくつかの手順の正確かつ複製可能な結果を得るための試金は重要で?...

腫瘍細胞の in vitroの侵襲と転移の可能性を評価する移動分析広く使用されます。よく、傷やスクラッチの試金は上皮シートのセルをオフにエリア1,2,3への移行を評価するために使用されます。スクラッチのアッセイを行う細胞が単層にメッキされ、ピペット チップ「スクラッチ」または無料のセル領域が作成されます。スクラッチの試金、一般的な培養装置を設定する簡単なウェル プレート、複数のサンプルの処理を可能にするうことができます。ただし、スクラッチでセル単一層から物理的に削除されます、細胞死をしばしば受けます。さらに、細胞外マトリックス プレートに取り付けがしばしばスクラッチ処理中に破損しています。同様に、シリコンの使用を挿入します (たとえば、Ibidi 室4) またはステンシルの5,6,7細胞の機械的崩壊とコーティングに使用されるマトリックス蛋白質の部分的な除去につながることができます、プレート。傷や傷の閉鎖を監視の試金の別の欠点は、スクラッチが閉じられるまで、細胞の移動を分析のみことができます彼らの期間限定コースです。
ドット アッセイを行うセルをコーティングしたまたはコーティング プレート8の上に円形のコロニーとしてめっき。このめっき方法の理論的根拠は、移行または挿入またはセルの削除によって文化を乱すことがなく細胞周辺に侵入できる定義されたエッジを持つ細胞シートを取得することです。ドット アッセイの全体的な目標は、移行細胞シート端変位または、同様高い時空間的解像度で細胞の渡り鳥の表現型を解析するタイムラプス イメージングを実行するコロニー径で測定したを観察することです。
細胞遊走はアイソレータケージ手がかり EGF などの成長因子、サイトカイン、ケモカインなどの様々 な影響を受けます。EGF が成長因子、その受容体は、EGF 受容体9へのバインディングを介してその生物学的効果を発揮し、腫瘍細胞4,9,10の侵襲と転移挙動が増加します。ドット試金を研究に使用するここで、EGF は、人間、侵襲性肺コロニー乳房癌細胞ライン (MCF10CA1a)8,11,12を形成細胞の移動を刺激します。

<table><tbody><tr><td>MCF10CA1a cells</td><td>Karmanos Research Institute</td><td>N/A</td><td>cells used for assay</td></tr><tr><td>mouse collagen IV</td><td>BD Biosciences</td><td>354233</td><td>used to coat plates</td></tr><tr><td>trypsin / EDTA</td><td>Thermo Fisher</td><td>25300054</td><td>used to remove cells from tissue culture plates</td></tr><tr><td>DPBS&amp;nbsp;</td><td>Mediatech</td><td>21-031-CV</td><td>used to wash cells</td></tr><tr><td>DMEM / F12</td><td>Thermo Fisher</td><td>11320082</td><td>used as culture medium</td></tr><tr><td>horse serum</td><td>Thermo Fisher</td><td>26050088</td><td>used to supplement culture medium</td></tr><tr><td>12 well glass bottom plate</td><td>MatTek</td><td>P12G-1.5-14-F</td><td>used to grow cells for imaging</td></tr><tr><td>EGF (epidermal growth factor, human recombinant)</td><td>Peprotech</td><td>AF-100-15</td><td>used to stimulate cells</td></tr><tr><td>fatty acid free BSA</td><td>Sigma</td><td>A8806-1G</td><td>used to make buffer for EGF</td></tr><tr><td>hematoxylin</td><td>Sigma</td><td>HHS32</td><td>used to stain colonies</td></tr><tr><td>eosin</td><td>Electron Microscopy Sciences</td><td>26051-10</td><td>used to stain colonies</td></tr><tr><td>37 &amp;#176;C, 5% CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Sanyo</td><td>model MCO-18AIC (UV)</td><td></td></tr><tr><td>Zeiss AxioObserver with incubator chamber and motorized stage</td><td>Zeiss</td><td>model Axio Observer.Z1</td><td>used for time lapse imaging</td></tr><tr><td>ORCA Flash LT sCMOS camera</td><td>BioVision</td><td>C11440-42U-KIT</td><td>used for timelapse imaging in combination with Zeiss AxioObserver</td></tr><tr><td>Metamorph</td><td>BioVision</td><td>MMACQMIC</td><td>software used for time lapse imaging</td></tr><tr><td>inverted microscope</td><td>Olympus</td><td>model IX70</td><td>used to count cells and to check colonies in tissue culture</td></tr><tr><td>plastic box</td><td>Lock&amp;amp;Lock</td><td>N/A</td><td>used as humid chamber (any tight closing plastic box that fits the plates can be used)</td></tr></tbody></table>

using the dot assay to analyze migration of cell sheets

複雑な生物は静的に表示が彼らの組織が継続的な売り上げ高の下です。細胞の年齢、死ぬとは新しい細胞に置き換えられて細胞移動組織内でしっかりと管弦楽に編曲された方法で。腫瘍の開発の間にこの平衡が妨げられるし、腫瘍細胞がローカル微小環境、遠いサイトに行き、最終的に遠隔部位に転移性の腫瘍を形成するために侵入するために原産の上皮を残します。ドットの試金はセル フリーエリアに細胞シートの網の動きを評価し、タイムラプス イメージングによる細胞遊走のパラメーターを分析する簡単な二次元の制約のない移行アッセイ。ドット アッセイは実証してここでは、侵襲的人間、肺コロニー形成乳房癌細胞ライン、MCF10CA1a を使用して上皮成長因子 (EGF)、乳癌細胞の悪性の可能性を高めるに知られている細胞の渡り鳥応答を分析して渡り鳥の細胞表現型を変更します。

侵襲型を使用して (図 1) を紹介ドット アッセイを行った肺コロニー モデル システムとして乳癌細胞 (MCF10CA1a) を形成します。ドット アッセイを生成するのに便利ですし、初心者の手でも再現性の高いセル ドットと簡単な試金 (図 1D) を実行します。ドット アッセイは、HE 染色、またはタイムラプス イメージ?...

Watch this Scientific Journal Video about Using the Dot Assay to Analyze Migration of Cell Sheets at JoVE.com

Using the Dot Assay to Analyze Migration of Cell Sheets

ドットの試金を使用して細胞シートの移行を分析するには

細胞遊走は開発、組織のメンテナンスと修復、および腫瘍発生の促進に欠かせない、成長因子、ケモカイン、サイトカインによって調節されています。このプロトコルでは、ドットの試金、アイソレータケージ キューに応答に接続されている、まとまりのある細胞シートの渡り鳥の表現型を評価するために二次元、制約のない移行アッセイについて説明します。

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

using-the-dot-assay-to-analyze-migration-of-cell-sheets

Research

JoVE Journal

Cancer Research

6.8K ビュー.  National Institutes of Health. 細胞遊走は開発、組織のメンテナンスと修復、および腫瘍発生の促進に欠かせない、成長因子、ケモカイン、サイトカインによって調節されています。このプロトコルでは、ドットの試金、アイソレータケージ キューに応答に接続されている、まとまりのある細胞シートの渡り鳥の表現型を評価するために二次元、制約のない移行アッセイについて説明?...

ビデオ: Using the Dot Assay to Analyze Migration of Cell Sheets

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

有害化学物質への曝露後の内皮細胞の遊走の評価

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

発生生物学

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

細胞浸潤および移行プロセスの評価: ビデオ顕微鏡ベースのスクラッチの比較創傷アッセイと boyden 教授区域の試金

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

がん研究

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

自動生細胞固体撮像素子を使用して単純な移行/侵略ワークフロー

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

微細加工チャネルにおける細胞移動の研究

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

生物工学

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

タンパク質機能に2DE  - ゲルスポットから：慢性リンパ性白血病で、HS1から学んだレッスン

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

医学

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

ヒト幹細胞由来内皮細胞およびGABAergicニューロンの移動、化学誘引、共培養アッセイ

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

神経科学

Quantifying Three-Dimensional Cell Migration Within and Into Granular Hydrogel Biomaterials

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for tissue integration. This article introduces two novel approaches for quantifying cell migration within and into granular hydrogels, highlighting the distinct applications of these scaffolds. First, a cell monolayer interface assay that simulates tissue growth into granular hydrogels for integration purposes is presented. Second, a spheroid-based assay is described, designed to track cell movement within the hydrogel matrix, specifically suited for applications involving cell delivery. Both methods enable precise and controlled measurements of cell migration, providing a comprehensive toolkit for researchers utilizing granular hydrogel scaffolds. The motivation for these methods stems from the need for tailored control over cell migration within the scaffold to align with specific applications. By optimizing and standardizing these quantification techniques, researchers can iteratively refine granular hydrogel properties, ensuring their effectiveness in diverse regenerative medicine contexts. This robust set of quantitative tools offers new opportunities to enhance granular hydrogel scaffolds, advancing their use in both cell delivery and tissue integration applications.

A protocol for the quantitative evaluation of 3D cell migration within and into the interface of granular hydrogels is presented here.

粒状ハイドロゲル生体材料内および粒状ハイドロゲル生体材料内への三次元細胞移動の定量化

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for ...

A Customizable Chamber for Measuring Cell Migration

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between cells, the extracellular matrix, and soluble and non-soluble chemical factors (e.g., chemoattractants). Standard methods for measuring the migration of cells, such as the Boyden chamber assay, work by counting cells on either side of a divider. These techniques are easy to use; however, they offer little geometric modification for different applications. In contrast, microfluidic devices can be used to observe cell migration with customizable concentration gradients of soluble factors1,2. However, methods for making microfluidics based assays can be difficult to learn.
Here, we describe an easy method for creating cell culture chambers to measure cell migration in response to chemical concentration gradients. Our cell migration chamber method can create different linear concentration gradients in order to study cell migration for a variety of applications. This method is relatively easy to use and is typically performed by undergraduate students.
The microchannel chamber was created by placing an acrylic insert in the shape of the final microchannel chamber well into a Petri dish. After this, poly(dimethylsiloxane) (PDMS) was poured on top of the insert. The PDMS was allowed to harden and then the insert was removed. This allowed for the creation of wells in any desired shape or size. Cells may be subsequently added to the microchannel chamber, and soluble agents can be added to one of the wells by soaking an agarose block in the desired agent. The agarose block is added to one of the wells, and time-lapse images can be taken of the microchannel chamber in order to quantify cell migration. Variations to this method can be made for a given application, making this method highly customizable.

This protocol details a customizable method to measure cell migration in response to chemoattractants that may also be used to determine the diffusion rate of a drug out of a polymer matrix.

細胞移動を測定するためのカスタマイズ可能な商工会議所

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between ...

生物学

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

細胞運動と関連するシグナル伝達事象の機械的制御を評価するための単細胞デュロタシスアッセイ

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...

The Transwell Migration Assay

Cells migration in response to chemical cues is crucial to development, immunity and disease states such as cancer. To quantify cell migration, a simple assay was developed in 1961 by Dr. Stephen Boyden, which is now known as the transwell migration assay or Boyden chamber assay. This set-up consists an insert which separates the wells of a multiwell plate into top and bottom compartments. Cells whose migration is to be studied are seeded into the top compartment and the chemoattractant solution is placed in the bottom compartment. After incubation, counting the cells in the bottom compartment allows quantification of migration induced by chemoattractants.
This video will review the commonly used experimental set-up for cell migration studies. Then we'll highlight a few key considerations, and outline a generalized protocol for running an experiment involving adherent cells. Lastly, we'll review various adaptations of this set-up currently being used to study different factors that affect migration.

The Transwell Migration Assay and Chemo-attractants

Transwell 移行アッセイ

Cells migration in response to chemical cues is crucial to development, immunity and disease states such as cancer.  To quantify cell migration, a simple ...