著者は、このプロジェクトのための資金を提供するためのクレムソン大学のクリエイティブ照会プログラムとNSF CBET1254609を認めます。

1.濃度勾配を作成するためにマイクロチャンネル発生室<ol><li> アクリル成形型部品を切削 <ol><li>所望の幅のアクリルの一部を取得します。典型的には、1/16インチ厚のアクリルシートを使用。より厚いシートは、それらが処理中に破損したり、ワープさせ、必要な機械的強度を持っていないだけでなく、非常に薄いシートをカットすることは困難です。 </li><li>ポリジメチルシロキサン（PDMS）内に空洞を生成するためにアクリル片の所望の形状のCADファイルを作成します。個々の実験プロトコルに関連する目的の勾配を達成するために、チャネル幅と長さを調整します。 <ol><li> 0.127センチメートル広いチャネル（0.254センチメートル長い）（ 図1）によって接続された2つの正方形（のx 0.254センチメートル0.254センチメートル）：ここでは、以下の寸法のアクリル作品を使用しています。 </li></ol></li><li>レーザー切断装置にCADファイルをインポートし、マヌーに応じてレーザーカッターでアクリルピースを配置facturerのプロトコル。 注：別の方法として、CAD設計から作品を3D-印刷します。この方法の利点は、代替材料が金型に使用することができることです。しかしながら、分解能および再現性は、レーザー切断に比べて三次元印刷に減少させることができます。 </li></ol></li></ol><img alt="図1" src="/files/ftp_upload/55264/55264fig1.jpg" /> 図1：マイクロチャンネル商工会議所のコンピュータ支援設計（CAD）表現。この画像は、マイクロチャネルのチャンバを作成するために必要なアクリルインサートのCAD図面を示します。 1 A）CAD図面、1B）インチに寸法とCAD図面の側面図、 図1C）インチに寸法とCAD図面の上面図の上面図<a href="http://ecsource.jove.com/files/ftp_upload/55264/55264fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <ol start="2"><li&gt;は、ポリジメチルシロキサン（PDMS）を注ぎます <ol><li>計量ボートでは、商業用エラストマー硬化剤（ 例えば 、シルガード194）1重量部で商業エラストマーベース（ 例えば 、シルガード184）10重量部を混合します。一般的に、エラストマー硬化剤の1グラムにエラストマーベースの10グラムを混ぜます。 </li><li> 5分間、マイクロピペットを用いて塩基及び硬化剤を組み合わせることかき混ぜます。 </li><li>真空ベルを設定し、閉じ込められた気泡を除去するために30分間真空中でPDMSと計量ボートを配置します。 </li><li>真空の電源を切り、計量ボートを削除します。小さなペトリ皿にアクリルカットアウトの上にPDMSを注ぎます。 PDMSが完全に挿入をカバーしていることを確認してください。 </li><li> PDMSは、室温で一晩（少なくとも18時間）を硬化させます。 </li></ol></li><li> PDMSからの挿入を削除します <ol><li> PDMSを硬化させた後、慎重にメスでアクリル作品の周りにPDMSを切断することにより、インサートを取り外します。 </li></ol&gt; </li></ol> 2.マイクロチャネルチャンバー内の細胞をプレーティング<ol><li> 細胞をプレーティングするためのチャンバを殺菌します。 注：PDMSは、エチレンオキシドまたは他の技術を用いて滅菌することができるが、迅速なUV処理は、迅速、安価、および細胞培養研究のために十分です。短いUV処理時間はPDMS片の構造又は機械的完全性を損ないませんでした。 <ol><li>標準的な細胞培養キャビネット内に、完全に70％エタノールを用いてチャンバを覆います。 </li><li>蓋をオフにして、層流フード内でペトリ皿を置きます。 </li><li>フードを終了し、UV光をオンにします。 1〜2時間UV光にさらします。 </li><li>層流フードを開き、流れを再確立するために15分を待ちます。 </li><li> 70％エタノールを削除します。 </li><li>滅菌リン酸緩衝生理食塩水（PBS）で2回チャンバを洗浄します。各洗浄のために1mLのPBSを使用してください。 PBSを削除し、室は蓋をオフにして一晩乾燥させます。 注：別の方法として、代わりに1が利用可能な場合、層流フードを使用しての、滅菌のためにUVチャンバーボックスを使用します。アルミホイルを敷いた段ボール箱の裏面にUV光源を配置することによって、UVボックスを作成します。アルミ箔は、サンプルの均一な照明を可能にするために光を反射します。 </li></ol></li><li> チャンバー内の細胞をプレーティング <ol><li>トリパンブルーと血球計数器を用いて細胞を数えます。 <ol><li> 0.4％トリパンブルーの400μLに100μLの細胞懸濁液を加え、穏やかに混合。静かにカバーガラスの下で両室を充填することにより、血球計数器に、この溶液100μLを適用します。 </li><li>血球計数器上のグリッドに注力して10X対物レンズで顕微鏡を使用してください。血球計数器上の16の正方形の1セット内生非染色細胞をカウントするために手の数取器を使用してください。 </li><li> 16の正方形のすべての4組のセルをカウントします。 16の正方形の4セットからの平均細胞数を取り、5×10 4を掛け。この最後の番号はnumbeです細胞懸濁液中の細胞/ mLのR。 </li></ol></li><li>各チャンバ内の1つのウェルに2,500細胞を追加します。これは〜3万/ cm 2の近コンフルエント細胞密度に変換されます。 </li><li>細胞は培地（ダルベッコ改変イーグル培地、10％ウシ胎児血清、1％ペニシリン - ストレプトマイシン）を加える前に、60分間室内に座ってできるようにします。 </li></ol></li></ol> 3.デキストラン浸しアガロースブロック<ol><li> アガロースブロックを作成します <ol><li> 3Dプリントモールド（2ミリメートル×2ミリメートル×2ミリメートル）にアガロース3％を注ぎます。 </li><li>アガロースは、任意の気泡を除去するために30分間、真空チャンバー内に固化することができます。 </li><li>金型からアガロースブロックを削除します。 注：別の方法として、2ミリメートルの厚さで小さなペトリ皿にアガロース3％を注ぎます。メスまたは他の切削工具を使用して、2ミリメートル×2ミリメートル×2ミリメートルのブロックをカット。これは、金型システムのように正確ではありませんが、それを行うのは少し楽にすることができます。 </li></ol></li><li>完全submerg走化性因子の所望の濃度の溶液中での電子アガロースブロック。濃度勾配の形成を評価するために、蛍光デキストランで第一室をフラッシュします。デキストランの濃度および分子量は、目的の可溶性因子または薬物のそれと一致する必要があります。 </li><li>アガロースブロック一夜を浸します。 </li></ol>可溶性因子の濃度勾配を評価するために、デキストラン拡散の4タイムラプス<ol><li>マイクロチャネル室の小さな正方形のウェルにデキストラン浸したアガロースブロックを配置します。 </li><li>セルイメージングシステム内のマイクロチャネル室を配置したり、タイムラプス機能を備えた別の蛍光顕微鏡を使用しています。製造元の指示に従ってタイムラプスを開始します。 注：示された結果は、GFPフィルター、50％の光、4月10日のpH、および4倍の倍率で撮影されています。 </li></ol>細胞増殖の5タイムラプス<ol><li>の一端にマイクロチャネルチャンバー内板細胞チャンバー。ここでは、3T3線維芽細胞を使用しています。マイクロチャネルチャンバー内で1つのウェルに2,500細胞を追加します。 2.2.1で説明したように、細胞をカウントします。 <ol><li>細胞は培地（ダルベッコ改変イーグル培地、10％ウシ胎児血清、1％ペニシリン - ストレプトマイシン）を加える前に、60分間室内に座ってできるようにします。 5％CO 2中37℃で播種した細胞とマイクロチャンネル室を保管してください。チャネルを介して細胞遊走を顕微鏡で見ることができます。 </li><li>マーカーを追加または距離セルフロントの動きを定量化するための出発点として機能するために、マーカーとしてチャンバ内の微小欠陥を使用します。 </li></ol></li><li>マイクロチャネルの一方の端部に集中し、成長因子、化学誘引物質、薬物、または他の因子が試験される（ここで、40％FBSを例として使用されている）を含むアガロースゲル（8 mm 3でブロック）を配置することによって勾配を確立しますチャンバー。 </li><li>移動セルフロントのタイムラプス画像を撮影します。ここでは、結果があちこちセルの画像を表示しますNTイメージングシステムを用いて12時間ごとに採取したもの。 </li><li>細胞増殖速度を定量化するために、セルの前面の写真を使用してください。最初のセルのフロント、チャネル壁、及び基準マーカによって定義される多角形のマスクを作成する（roipoly）MATLABの関数polyfitを使用して、増殖速度を計算します。このマスクの寸法は、平均セルフロント速度が算出されたフロントの移動距離をもたらします。他の研究では、同様の方法8を利用しています。 注：同じ距離で可溶性因子の濃度になるように、各フレーム上の細胞増殖前面の縁を比較してください。可溶性因子の濃度勾配は、1次元拡散モデルの近似を用いて計算されます。 </li><li>ファロイジンおよびDAPI染色を用いて細胞の蛍光画像を撮ります。 <ol><li>ファロイジンで染色する前に細胞を固定。 <ol><li> 37℃で温め、4％パラホルムアルデヒド。 </li><li>セルをカバーするのに十分な4％パラホルムアルデヒドを加えます。で細胞を保ちます10分間パラホルムアルデヒド。 </li><li> 15分間、PBSで2回、それぞれの時間をすすぎます。セルをカバーするのに十分なPBSを使用してください。 </li></ol></li><li>細胞からPBSを除去し、細胞をカバーするのに十分な透過化溶液（PBS中0.1％トリトンX-100）を追加します。 10分間この溶液を残します。 </li><li>毎回5分間、PBSで2回洗浄します。セルをカバーするのに十分なPBSを使用してください。 </li><li>細胞からPBSを除去し、20分間溶液（PBS中の1％ウシ血清アルブミン）でブロッキング加えます。 </li><li>ブロッキング溶液を除去し、5分間PBSで各回2回洗浄します。セルをカバーするのに十分なPBSを使用してください。 </li><li>使用されていない場合、この時点から、アルミニウム箔で光から細胞を保護します。細胞からPBSを削除し、1時間、PBSで希釈したファロイジン488 1:50に追加します。 </li><li>各洗浄のための5分間、PBS中で2回洗浄します。セルをカバーするのに十分なPBSを使用してください。次に、細胞からPBSを削除します。 </li><li> 15分間、細胞をPBS 1,000：DAPI（4 &#39;、6-ジアミジノ-2-フェニルインドール）を追加し、1に希釈しました。 </li><li> DAPIを削除し、各洗浄のための5分間PBSで2回細胞を洗浄します。 </li><li>最後の洗浄を削除し、セルをカバーするのに十分なPBSを追加します。細胞は、現在、蛍光顕微鏡で画像化することができます。 </li></ol></li></ol>

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著者らは、開示することは何もありません。

我々のマイクロチャネル室は、成長因子及び化学誘引物質に応答した細胞遊走の速度を決定し、ポリマーマトリックスからの薬物の拡散速度を測定することを含む、目的の多数のために使用することができます。細胞を増殖し、チャンバの一端に化学誘引物質を配置するために我々のマイクロチャネル室を利用することが可能です。細胞は、化学誘引物質に応答して成長し、細胞遊走、細胞?...

In order for vital processes such as wound healing, immune responses, and embryonic development to occur, cell migration must take place. Cell migration involves the interaction between cells and neighboring cells, the extracellular matrix, and soluble chemical cues (attractants or repellants). As an example, in the process of wound healing, fibroblasts play an integral role in fibrogenesis and wound contraction, where the cells are recruited to the site of injury to synthesize collagen in order to form the extracellular matrix3. Numerous mechanisms behind the migration of fibroblasts to a wound site have been studied, and they include different mechanical, physical, electrical, and chemotactic factors4. Fibroblasts respond especially well to different concentration gradients of growth factors. These different growth factors work together to optimize tissue regeneration5. While observing the chemotactic response of fibroblasts to growth factor concentrations, one can study the pattern of directional migration of fibroblasts and how they orient themselves around physical obstacles in order to reach their destination. Therefore, the goals of this study were to first develop a system in which fibroblast growth could be tracked under guidance by physical barriers and to secondly model the growth of fibroblasts as they navigate through the system.
Currently, the Boyden chamber assay is the most widely used system to measure the migration of cells6. The Boyden chamber consists of a two-chamber multi-well plate where each well may contain medium with or without chemoattractants7. A filter membrane provides a porous interface between the two chambers in each well; this creates a barrier so that cells cannot pass through unless it is by active migration. Typically, for the Boyden chamber, a chemoattractant is added to the lower chamber, and the system is allowed to equilibrate to form a gradient between the upper and lower wells4. One problem with the Boyden chamber assay is that steep gradients end up forming along a single axis perpendicular with the surface of the membrane. This causes the difference in the chemoattractant concentration between the upper and lower wells to be a lower than what was originally expected. Due to this constraint, the Boyden chamber assay makes it hard to correlate specific cell responses with particular gradient characteristics, such as the slope and the concentration difference. Without these measurements, it is hard to study multi-gradient signal integration.
To address some of the constraints of the traditional Boyden chamber assay, microfluidic assays have been developed to form customizable concentration gradients1. Standard methods for creating microfluidic systems require clean rooms for lithographic techniques. These techniques can be difficult to learn especially in a standard classroom setting. Thus, we have designed a chamber system for measuring cell migration that can be made without using a clean room. Using our system, the wells of the assay can be adjusted to a preferred size, and a linear concentration gradient of customized slope can be produced. This allows for accurate measurement of chemotaxis from random movement. The design is an inexpensive and easy-to-use system to model cell growth in response to different chemical stimuli.

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			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Sigma-Aldrich</td>
			<td>761036</td>
			<td>poly(dimethylsiloxane) 2-part kit including silicone elastomer base and silicone elastomer curing agent</td>
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		<tr>
			<td>Acrylic Sheets</td>
			<td>US Plastic</td>
			<td>44200</td>
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			<td>Disposable Petri Dishes&#160;</td>
			<td>Falcon&#160;</td>
			<td>25373-041</td>
			<td></td>
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			<td>Fluorescein isothiocyanate&#8211;dextran</td>
			<td>Sigma-Aldrich</td>
			<td>FD20s-100MG</td>
			<td></td>
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		<tr>
			<td>Agarose, Type I, Low EEO</td>
			<td>Sigma-Aldrich</td>
			<td>A6013-100G</td>
			<td></td>
		</tr>
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			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Fisher Scientific</td>
			<td>11965092</td>
			<td>Cell media components</td>
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			<td>Fetal Bovine Serium</td>
			<td>Fisher Scientific</td>
			<td>16000036</td>
			<td>Cell media components</td>
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			<td>Penicillin-streptomycin</td>
			<td>Fisher Scientific</td>
			<td>15140148</td>
			<td>Cell media components</td>
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			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BP24384</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS XL Cell Imaging System</td>
			<td>Thermo Fisher Scientific</td>
			<td>AME3300</td>
			<td>Instrument used for taking time-lapse images</td>
		</tr>
		<tr>
			<td>Versa LASER</td>
			<td>Universal Laser Systems, Inc.&#160;</td>
			<td>Model number VLS2.30</td>
			<td>Laser cutter used for cutting plastic</td>
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a customizable chamber for measuring cell migration

細胞遊走は免疫応答、成長、および創傷治癒の重要な一部です。細胞遊走は、細胞外マトリックス、および可溶性および非水溶性の化学的因子（ 例えば 、化学誘引物質）との間の相互作用が関与する複雑なプロセスです。このようなボイデンチャンバーアッセイ、分周器のいずれかの側に細胞を計数することにより、作業のような細胞の遊走を測定するための標準的な方法。これらの技術は、使いやすいです。しかし、彼らは、異なるアプリケーションのために少しの幾何学的修正を提供しています。対照的に、マイクロ流体デバイスは、可溶性因子1、2のカスタマイズ可能な濃度勾配を有する細胞遊走を観察するために使用することができます。しかし、マイクロ流体ベースのアッセイを作製するための方法は、学ぶことは困難な場合があります。 ここでは、化学物質の濃度勾配に応答して細胞遊走を測定するための細胞培養チャンバを作成するための容易な方法を記載します。私たちの細胞マイルグレーションチャンバー法は、様々な用途のための細胞移動を研究するために、別の直線濃度勾配を作成することができます。この方法は、使用が比較的容易であり、典型的には学部学生によって行われます。 マイクロチャンネル室はよくペトリ皿に、最終的なマイクロ流路室の形状のアクリルインサートを配置することによって作成されました。この後、ポリ（ジメチルシロキサン）（PDMS）は、インサートの上に注ぎました。 PDMSを硬化させ、次いで、インサートを除去しました。これは、任意の所望の形状や大きさのウェルの作成を可能にしました。細胞は続いてマイクロチャネルチャンバーに添加することができる、可溶性薬剤は、所望の薬剤にアガロースブロックを浸漬することによってウェルのいずれかに添加することができます。アガロースブロックをウェルのいずれかに添加し、タイムラプス画像は、細胞遊走を定量するために、マイクロチャネル室を撮影することができます。この方法のバリエーションは、このメソッドのHIGを作り、所与の用途のために作ることができますHLYカスタマイズ可能。

図2は、細胞がメッキされているところから、チャネルの反対側の端部に配置されたウシ胎児血清の勾配に応じて、チャネル全体のセルフロントの動きを示しています。細胞フロント48時間（ 図2A）、72時間（ 図2B）、96時間（ 図2C）、および120時間（ 図2D）ポストプレーティングで示されています。セル?...

Watch this Scientific Journal Video about A Customizable Chamber for Measuring Cell Migration at JoVE.com

A Customizable Chamber for Measuring Cell Migration

このプロトコルはまた、ポリマーマトリックスからの薬物の拡散速度を決定するために使用される化学誘引物質に応答した細胞遊走を測定するためのカスタマイズ可能な方法を詳述します。

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 ...

a-customizable-chamber-for-measuring-cell-migration

Research

JoVE Journal

Biology

10.5K ビュー.  Clemson University. このプロトコルはまた、ポリマーマトリックスからの薬物の拡散速度を決定するために使用される化学誘引物質に応答した細胞遊走を測定するためのカスタマイズ可能な方法を詳述します。 

ビデオ: A Customizable Chamber for Measuring Cell Migration

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

FMラベリングを用いた神経細胞の測定エキソサイトーシス

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

生物学

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The two-level microfluidic device is fabricated using a two-step standard negative resist lithography process 1. The first level contains microchannels with inlet and outlet ports at each end. The second level contains microscale circular holes located midway of the channel length and centered along with channel width. Passive pumping method is used to pump fluids from the inlet port to the outlet port 2. The microfluidic device is integrated with off-the-shelf perfusion chambers and allows seamless integration with the electrophysiology setup. The fluids introduced at the inlet ports flow through the microchannels towards the outlet ports and also escape through the circular openings located on top of the microchannels into the bath of the perfusion. Thus the bottom surface of the brain slice placed in the perfusion chamber bath and above the microfluidic device can be exposed with different neurotransmitters. The microscale thickness of the microfluidic device and the transparent nature of the materials [glass coverslip and PDMS (polydimethylsiloxane)] used to make the microfluidic device allow microscopy of the brain slice. The microfluidic device allows modulation (both spatial and temporal) of the chemical stimuli introduced to the brain slice microenvironments.

We demonstrate fabrication of a simple microfluidic device that can be integrated with standard electrophysiology setups to expose microscale surfaces of a brain slice in a well controlled manner to different neurotransmitters.

マイクロ流体ネットワークを用いた脳スライスの刺激と標準灌流チャンバー

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

ショウジョウバエの眼成虫ディスクに付属してグリア細胞の移行のイメージングを生きる

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

光トラップを用いた細菌細胞の曲げ剛性の測定

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

の活性を測定するアッセイ大腸菌誘導性リジンDecarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

末梢血幹細胞移動の解析のためのマウス精巣挙筋で微小循環の生体顕微鏡

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

セル通信解析のための単一細胞のマイクロインジェクション

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

インビトロでの細胞遊走および浸潤アッセイにおける

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

中間径フィラメントと微小管 2次元直接確率光再構成顕微鏡イメージング

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.