仕事は、私たちの研究室は、国立衛生研究所からの助成金によってサポートされています。

準備<ul><li>カバースリップはオートクレーブする必要があります。 </li><li>滅菌蒸留水または脱イオン水は、溶液を調製し、洗浄のカバースリップのために使用する必要があります。 </li><li> AC（40％w / v）とビス- AC（1％w / v）のソリューションは、0.2μmのろ過滅菌されています。使用直前および滅菌フィルターの前に、10％過硫酸アンモニウム（100μg/ml水APS）を準備する。毎月のAPSソリューションを交換してください。 </li><li>オートクレーブできないような3 - APTMS、クロロホルム、glutaradehyde、NHS、およびSurfaSilなどの化学試薬は、ヒドロゲルの調製のためにのみ使用される割り当てられた瓶に保管されています。 </li><li>最良の結果を得るには、ハイドロゲルは、適切なECM​​蛋白質との一晩のインキュベーションの後数日以内に利用されるべきである。 </li><li>ハイドロゲルの注ぐ前に50ミリリットルポリプロピレンファルコンチューブにクロロホルム（10mlは通常十分なトップ20カバースリップ用です）の10％SurfaSilの溶液を調製します。 （私たちの研究室は、通常0.5％のグルタルアルデヒドのインキュベーションステップの間にトップカバースリップをsiliconizes）。少なくとも10分間ファルコンチューブやロックにカバースリップを追加。 SurfaSilソリューションをデカントし、空気は、ヒドロゲルが準備される生物学的安全キャビネットのキムワイプでカバーグラスを乾燥させる。 </li><li>次のように熱不活化BSA溶液を調製します：PBSの脂肪酸フリーBSAの20 mg / mlの溶液を30分間68℃水浴中でインキュベートされる。次いで、この溶液を滅菌濾過し、4℃で保存されています</li></ul>手順<ol><li> 150 mmのペトリ皿の下半分にパラフィルムの層を配置。 </li><li>パラフィルムの上に9 25 mmのカバースリップするまで置き、1 mlの0.1 M NaOHでそれらをカバー。 3分間インキュベートし、次いで真空ラインと吸引。 </li><li>各カバースリップ上の場所0.5ミリリットルの3 APTMS、化学フードでの作業。 3分間インキュベートし、APTMSを吸引除去する。ここで時間をおきすぎている場合、泡が形成されます。 </li><li>同じ皿で20 mLの脱イオン水で一度カバーグラスを洗浄します。曲がったピンセットを使って皿からカバーガラスを取り外し、それらの処理側が新たな150 mmディッシュに、上向きにし、それらを転送する。 10分ごとに洗浄するため、ロッカーに、脱イオン水で3回カバーグラスを洗ってください。あなたがすべてAPTMSを削除できなかった場合、それは次のステップでグルタルアルデヒドと反応し、白色の濁った沈殿物（図1）のままになります。 </li><li>グルタルアルデヒド（ご使用の前〜10分）を解凍。 </li><li>湾曲鉗子を使用して、パラフィルムで積層しクリーンな皿にカバースリップを転送し、残り​​の液体を吸引除去する。必要に応じて残りの水をブロットに真空ラインまたはキムワイプを使用してください。 </li><li>滅菌脱イオン水で0.5％グルタルアルデヒド0.5mlを完全にそれぞれのカバーをカバーし、化学フード中で30分間インキュベートする。グルタルアルデヒドを吸引除去する。リンスと手順4のようにカバーグラスを洗う。完全にカバーグラスを乾燥させます。[あなたが乾燥した地域でここに停止し、数週間のためにカバースリップを残すことができます]。 </li><li>あなたがハイドロゲルを準備する準備ができたら、生物学的安全キャビネットの表面にテープで留められているパラフィルムのシートに、カバーガラス、反応性側を転送するために湾曲したピンセットを使用してください。カバーガラスは、パラフィルムの表面に平らになっていることを確認します。 </li><li>トルエンの飽和NHS溶液を準備します。 （特定の実験のための十分なトルエンでNHSの少量を溶かす。NHSはもはや溶解しなくなるまでビットによってNHSのビットを追加していきます。飽和溶液は、通常、曇りとピンクです。） </li><li>次に、目的のアクリルアミドの割合に到達するアクリルアミド、ビスアクリルアミド、水とAPSを準備。 0.8ミリリットルの合計体積ため、以下に示すようにマイクロチューブ内の試薬を加える。 </li><li>その後、一度に分注し、NHSを追加し、0.8ミリリットルのACソリューションにTEMED、渦は簡単に、すぐに生物学的安全性のフードでカバースリップあたり140μlを使用して3から5のゲルを注ぐ。可能な限り、この点からすべてのステップは、生物学的安全キャビネット内で実行する必要があります。 [注：異なるサイズのカバースリップは必要性に基づいて利用することができます。直径18mmのカバースリップが使用されている場合は、〜33カバースリップ当りμlのACのソリューションと18 mmのトップシリコーンカバースリップを使用してください。] <table border="1"><tbody><tr><td></td><td colspan="4">ビス- AC（％） </td></tr><tr><td></td><td> 0.3（硬い） </td><td> 0.15 </td><td> 0.06 </td><td> 0.03（ソフト） </td></tr><tr><td></td><td> μL </td><td> μL </td><td> μL </td><td> μL </td></tr><tr><td>水</td><td> 402 </td><td> 522 </td><td> 594 </td><td> 618 </td></tr><tr><td> AC </td><td> 150 </td><td> 150 </td><td> 150 </td><td> 150 </td></tr><tr><td>ビス- AC </td><td> 240 </td><td> 120 </td><td> 48 </td><td> 24 </td></tr><tr><td> APS </td><td> 8 </td><td> 8 </td><td> 8 </td><td> 8 </td></tr><tr><td> TEMED</td><td> 1 </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td> NHS </td><td> 228 </td><td> 228 </td><td> 228 </td><td> 228 </td></tr></tbody></table></li><li>それが重合を開始する前に迅速に各ゲルの上にシリコーン処理25 mmのカバースリップを配置。トップカバーの加算は、ACが完全に底カバースリップをカバーできるようにする必要があります。 ACが重合するまで室温でこの&quot;サンドイッチ&quot;をインキュベートする。 （重合がいつ発生したかを確認するためにマイクロ遠心チューブ中の残留ACのソリューションを確認してください。柔らかいもののための硬いゲルと少し長めのため通常数分で十分です。）。 </li><li>慎重にサンドイッチを拾う（滅菌手袋で！）、それがオーバーハング重合ゲルをするまで、上部カバーを外します。その後、ゲルを離れてそれを取り外すことができます。あなたがトップカバーを取り外す前に、あまりにも長く待つ場合、カバースリップが削除される場合は、ゲルがリッピングされます。 </li><li>トップカバーを捨てる。ウェル2 mlのPBS / 6ウェルプレートに底ゲルカバースリップを（以下、ハイドロゲルと呼ばれる）に置きます。 PBSを各ウェルにゲルカバースリップの前または後に添加することができます。ロッカー、洗濯ごとに5分で、PBSで3回ヒドロゲルを洗ってください。 </li><li>ヒドロゲルの必要数が準備されるまで繰り返します11-14繰り返します。 </li><li>フィブロネクチン溶液（PBS中の3μg/ mlの）または他のECMタンパク質（Kleinらを参照してください。詳細については13。）2mlで各ハイドロゲルをカバー。 ECMタンパク質が共有結合し、4℃で一晩インキュベーション中にハイドロゲルにバインドするには</li><li> 37℃最低℃で30分間インキュベーター内でCのための無血清培地に1mg/mlの熱不活性化脂肪酸フリーBSAでNHSを未反応のECMソリューションとブロックを吸引除去する。滅菌PBSまたは細胞培養培地で1回ヒドロゲルをすすぎます。 </li><li> FBSを含む適切な培養培地で細胞をプレート。ハイドロゲル上に播種する細胞の数は、実験に必要な拡散細胞とコンフルエントの度合いに基づいて、ユーザによって決定されるべきである。約10 5細胞は通常、ウェスタンブロットおよび定量PCR分析のために十分です。 </li><li>インキュベーション期間後、細胞のタンパク質またはmRNAを抽出することができます。カバースリップを慎重に曲がったピンセットを使用してウェルから除去し、実験台上にパラフィルムのシートに沿って（2〜3センチメートル）間隔をあけられているの溶解緩衝液100μlの液滴の上に（下にセル側）に配置されます。 （私たちは、ウェスタンブロッティングや定量PCR用RNAを分離するためのサンプルを準備するために、それぞれ、標準のSDSサンプルバッファーとTRIZOLを使用する。）正確に1分間溶解バッファーで細胞をインキュベートする。カバースリップを削除し、マイクロ遠心チューブに溶解用緩衝液を移す。また唯一のRNA抽出のために、ユーザーは新しい6ウェルプレートに各ハイドロゲルを転送し、TRIZOL /ウェルの1mlを追加することができます。 3分間インキュベートし、マイクロ遠心チューブに保存するためにトリゾールソリューションを削除する。 </li></ol>このような免疫蛍光、BrdU染色などの手順に関する追加情報は、ハイドロゲル上に播種細胞用トランスフェクション、等はKleinらに記載されています。 2007 13。 代表的な結果 APTMSの添加後、カバースリップの十分な洗浄は、&quot;反応&quot;カバーグラスを製造するのに重要なステップです。いずれかの完全にAPTMSを削除するには失敗した場合は、次の手順でグルタルアルデヒドと反応して、図1Aに示すように白い曇りの沈殿物が生成されます。図1Bは、適切に洗浄し、乾燥カバースリップを示しています。沈殿物が生じた場合はカバースリップは、使用できなくなったとして、全体の手順は、最初から再起動する必要があります。 ECMタンパク質とのハイドロゲルの形成とコーティングした後、一晩かけて、細胞は翌日播種することができます。図2に示すように、硬い対柔らかいヒドロゲルに拡散セルの間に明確な違いがあります。マウス胚性繊維芽細胞（MEF）のファロイジン染色で見られるようにソフトなハイドロゲルと比較して、細胞が硬い上に大きい程度に広がった。確かに、ソフトなハイドロゲルに取り付ける部分の細胞は、コンパクトなままとなり、少ない効率的に添付してください。 唯一のMEFの形態を図2に示されているが、拡散セル内の差は11-12,14をテストしたいくつかの他の細胞株に共通したものです。 成功の秘訣<ol><li>手続きが開始されると、それは&quot;反応性&quot;側にカバースリップを維持し、コーティングされている側の心に留めておくことは非常に重要です。 </li><li>手袋はできるだけ無菌である作業環境を提供するために、手順の間にすべての時に着用してください。 </li><li>最初の手順のほとんどは、化学煙フード、実験室間で実行されます。カバーガラスから余分なグルタルアルデヒドを除去する以後のすべてのステップは、生物学的安全キャビネットの下で実行する必要があります。 </li><li>セルspreaの違いによる鼎（図2）、我々は硬いハイドロゲルと比較して柔らかいハイドロゲル上の細胞数の2倍のシードをお勧めします。 </li><li> ACの重合プロセスは非常に速いです。初めてご利用の場合、一つはAPSの量を減らすことができますまたは重合プロセスを延長するソリューションにTEMED。一度に少数のカバースリップ以上を準備しようとしないでください。 </li><li> G0での同期が必要とされる細胞周期の研究において、我々は通常血清飢餓細胞をプラスチック製の培養皿に、細胞をトリプシン処理し、マイトジェン（FBSおよび/または増殖因子）の存在下でさまざまなコンプライアンスはのヒドロゲルにそれらを再シード。この手順では、G0 -同期細胞の出発個体群はすべての試料において同一であることを保証します。しかし、多くのシグナル伝達の研究のために、必要なマイトジェン刺激期間は、細胞の付着と拡散を可能にするために十分な長さではありません。このケースでは、ヒドロゲルの細胞を血清飢餓とし、直接マイトジェンでそれらを刺激する。 </li></ol><img src="/files/ftp_upload/2089/2089fig1a.jpg" alt="図1a" /> 図1A。不適切にカバースリップを洗浄した。APTMSを添加した後、カバースリップは、グルタルアルデヒド溶液の添加の前に1〜2分間洗浄した。沈殿物は、手順で説明した手順に従って洗浄されていないカバースリップ上に形成される。 <img src="/files/ftp_upload/2089/2089fig1b.jpg" alt="図1b" /> 図1B。適切にカバースリップを洗浄した。APTMSを添加した後、カバースリップは10分ごとにグルタルアルデヒド溶液の添加前に3回洗浄した。いいえ沈殿物はカバースリップに形成しない。 <img src="/files/ftp_upload/2089/2089fig2.jpg" alt="図2" /> 図2。剛性の異なるハイドロゲル上での細胞の形態。MEFSを設立は9時間のための高または低剛性のフィブロネクチンでコーティングされたハイドロゲル上に播種した。インキュベーション期間後、細胞は、固定透過性およびF -アクチンに結合するFITC -ファロイジンで染色した。高剛性ゲル上でMEFは、低剛性のハイドロゲル上に播種に比べてストレスファイバーを展示し、よく分散されています。スケールバー=50μmの。 <img src="/files/ftp_upload/2089/2089fig3.jpg" alt="図3" /> 図3。マウスのサイクリンD1のmRNAレベルの代表的な定量的PCRの結果。血清飢餓マウス胚線維芽細胞は、（0.3％アクリルアミド）高い（3％アクリルアミド）や低剛性のハイドロゲル上に播種し、9時間を10％FBSで刺激した。 RNAの抽出に続いて、リアルタイム定量PCR解析は、サイクリンD1のmRNAレベル（18S RNAに正規化）を行った。 G0は静止細胞からサイクリンD1 mRNAを表します。サイクリンD1のmRNAレベルは有意に硬いヒドロゲル上ではなく、柔らかいヒドロゲルに増加する。 SD重複PCR反応の - データは、+ /を意味している。

<ol>
	<li>Beattie, D., Xu, C., Vito, R., Glagov, S., Whang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+analysis+of+heterogeneous,+atherosclerotic+human+aorta.">Mechanical analysis of heterogeneous, atherosclerotic human aorta.</a> J Biomech Eng. 120, 602-607 (1998).</li><li>Bernini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+stiffness,+intima-media+thickness+and+carotid+artery+fibrosis+in+patients+with+primary+aldosteronism.">Arterial stiffness, intima-media thickness and carotid artery fibrosis in patients with primary aldosteronism.</a> J Hypertens. 26, 2399-2405 (2008).</li><li>Boonyasirinant, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+stiffness+is+increased+in+hypertrophic+cardiomyopathy+with+myocardial+fibrosis:+novel+insights+in+vascular+function+from+magnetic+resonance+imaging.">Aortic stiffness is increased in hypertrophic cardiomyopathy with myocardial fibrosis: novel insights in vascular function from magnetic resonance imaging.</a> J Am Coll Cardiol. 54, 255-2562 (2009).</li><li>Discher, D. E., Janmey, P., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+cells+feel+and+respond+to+the+stiffness+of+their+substrate.">Tissue cells feel and respond to the stiffness of their substrate.</a> Science. 310, 1139-1143 (2005).</li><li>Duprez, D. A., Cohn, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+stiffness+as+a+risk+factor+for+coronary+atherosclerosis.">Arterial stiffness as a risk factor for coronary atherosclerosis.</a> Curr Atheroscler Rep. 9, 139-144 (2007).</li><li>Lee, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+mechanical+properties+of+human+atherosclerotic+tissue+by+high-frequency+intravascular+ultrasound+imaging.+An+in+vitro+study.">Prediction of mechanical properties of human atherosclerotic tissue by high-frequency intravascular ultrasound imaging. An in vitro study.</a> Arterioscler Thromb. 12, 1-5 (1992).</li><li>Levental, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling.">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139, 891-906 (2009).</li><li>Paszek, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8, 241-254 (2005).</li><li>Samani, A., Zubovits, J., Plewes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+moduli+of+normal+and+pathological+human+breast+tissues:+an+inversion-technique-based+investigation+of+169+samples.">Elastic moduli of normal and pathological human breast tissues: an inversion-technique-based investigation of 169 samples.</a> Phys Med Biol. 52, 1565-1576 (2007).</li><li>Wells, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+matrix+stiffness+in+regulating+cell+behavior.">The role of matrix stiffness in regulating cell behavior.</a> Hepatology. 47, 1394-1400 (2008).</li><li>Pelham, R. J., Wang, Y. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+locomotion+and+focal+adhesions+are+regulated+by+substrate+flexibility.">Cell locomotion and focal adhesions are regulated by substrate flexibility.</a> Proc. Natl. Acad Sci USA. 94, 13661-13665 (1997).</li><li>Klein, E. A., Yung, Y., Castagnino, P., Kothapalli, D., Assoian, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion,+cellular+tension,+and+cell+cycle+control.">Cell adhesion, cellular tension, and cell cycle control.</a> Methods Enzymol. 426, 155-175 (2007).</li><li>Klein, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cycle+control+by+physiological+matrix+elasticity+and+in+vivo+tissue+stiffening.">Cell-cycle control by physiological matrix elasticity and in vivo tissue stiffening.</a> Current Biology. 19, 1511-1518 (2009).</li></ol>

利害の衝突は宣言されません。

ハイドロゲルの重合プロセスの重要な要素は、細胞がガラスのカバースリップではなく、ECMコーティングされたハイドロゲル自体にバインドできるようにする気泡の形成を避けるためです。これは、慎重にボルテックスし、視覚的に気泡がゲル中に閉じ込められたとなっていないことを確認した後、重合溶液をピペッティングすることによって防ぐことができます。私たちは、常に実験のた?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Glutaraldehyde, 70%</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G7776</td>
<td>Store at -20&deg;C</td>
</tr>
<tr>
<td>3-APTMS (3-Aminopropyltrimethosysilane 97%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>281778</td>
<td>Store at room temperature</td>
</tr>
<tr>
<td>SurfaSil Siliconizing Fluid</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>42800</td>
<td>Store at room temperature</td>
</tr>
<tr>
<td>NHS (N-hydroxysucinimide Ester)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A-8060</td>
<td>Store at 4&deg;C Replace monthly</td>
</tr>
<tr>
<td>Albumin, bovine serum, essentially fatty acid free</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A6003-100G</td>
<td>Store at 4&deg;C</td>
</tr>
<tr>
<td>Coverslips (25mm)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-86 25 Cir 1D</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips (18mm)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-84 18 Cir 1D</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

studying the effects of matrix stiffness on cellular function using acrylamide-based hydrogels

組織の剛性は、細胞機能の重要な決定因子であり、組織の硬さの変化は、一般的に線維症、癌、心血管疾患1月11日に関連付けられています。細胞機能を研究するために、従来の細胞生物学的なアプローチは、弾性ECMや組織間のECMの剛性の変動の影響を考慮することはできません堅い基層上の培養細胞を（プラスチック製の皿やガラスのカバースリップ）を伴います。 in vitroでの生体組織のコンプライアンスの条件でモデルに、我々と他のECMコーティングされたハイドロゲルを使用してください。当研究室では、ヒドロゲルは生物学的に12見た組織のコンプライアンスはの範囲を模倣することができるポリアクリルアミドに基づいています。 &quot;リアクティブ&quot;カバースリップは3 APTMSを加えて水酸化ナトリウムとのインキュベーションによって生成されます。グルタルアルデヒドは、3 APTMSとポリアクリルアミドゲルを架橋するために使用されます。アクリルアミド（AC）、ビスアクリルアミド（ビス- AC）および過硫酸アンモニウムの溶液をハイドロゲルの重合に使用されます。 N -ヒドロキシスクシンイミド（NHS）は、ハイドロゲルに架橋ECMタンパク質にACのソリューションに組み込まれています。ハイドロゲルの重合に続いて、ゲルの表面は、フィブロネクチン、ビトロネクチン、コラーゲンなどの選択肢のECM蛋白質で被覆されているハイドロゲルの剛性は、レオロジーや原子間力顕微鏡（AFM）によって決定され、溶液12にACおよび/ ​​またはビス- ACの割合を変えることによって調整することができます。このように、基層の剛性は、レオロジーやAFMを用いて定量することができる生体組織の硬さに一致させることができます。次いで、細胞をこれらのヒドロゲルに播種し、必要な実験条件に基づいて培養することができる。分子解析のための細胞とその回復のイメージングは​​簡単です。この記事では、我々は20,000パスカル&lt;3000パスカルと硬い基層/組織Eを持つものとして&gt;弾性率（E）を有するものとして、ソフト基層を定義します。

Watch this Scientific Journal Video about Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels at JoVE.com

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

細胞機能上の基層の剛性の影響をモデル化することができます<em&gt; in vitroで</em&gt;様々なコンプライアンスはのポリアクリルアミドハイドロゲルを使用。

アクリルアミド系ハイドロゲルを用いた細胞機能のマトリックスの剛性の影響を研究

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

studying-effects-matrix-stiffness-on-cellular-function-using

Research

JoVE Journal

Biology

21.9K ビュー.  University of Pennsylvania. 細胞機能上の基層の剛性の影響をモデル化することができます in vitroで様々なコンプライアンスはのポリアクリルアミドハイドロゲルを使用。

ビデオ: Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

組織工学のための3Dハイドロゲルの細胞のカプセル化

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

生物学

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 ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

内因性野生型BRCA1を発現する細胞を用いて相同組換えにBRCA1の変異の影響を識別

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

偏上皮細胞へのプラスミドのマイクロインジェクションによる低分子量GTPaseの機能を解析する

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

シトクロムを使用してミトコンドリア呼吸機能の可視化 Cオキシダーゼ/コハク酸脱水素酵素（COX / SDH）二重標識組織化学

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

に低酸素の影響を検討するために定義され気体環境を作成する<em&gt; C.エレガンス</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

上の細菌の影響を測定する<em&gt; C.エレガンス</em卵保持アッセイを用いて&gt;行動

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

ゼブラフィッシュ ランゲルハンス島における単一セルの解像度カルシウム イメージングを用いた β-細胞機能の解析

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

タンパク質とマイクロRNA変調がアディポサイト機能に及ぼす影響を研究するアディポサイト細胞培養モデル

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

イン・ヴィトロ E3ユビキチンリガーゼ関数の解析

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...