作者は感謝したいと思います：博士フランク·江、デビッド·リン博士バーナードYurke博士ウダイChippadaをDNAゲル技術の開発に貢献するために、博士ノレルHadzimichalis、スミット·シャー、キンバリーピーターマン、ロバートArterこの原稿の彼らのコメントや編集をするため;脊髄研究に関するニュージャージー委員会（助成＃07A-019-SCR1、NAL）とニュージャージー神経科学研究所（MLP）を含む資金調達源; 図3を転載する許可を図2と図4およびバイオマテリアルを転載する許可のための組織工学のパブリッシャー、パートA。

<ol>
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著者らは、開示することは何もない。

細胞接着は、それらの細胞機能に対する動的な組織の剛性の役割を研究するための理想的なモデルとなる前と後の軟化または硬くするDNAゲルの能力。すべての3つの設計では、機械的および生物学的研究に使用されている。しかし、すべての3つのデザインは、架橋の長さはDNAゲル弾性（ 表2）影響を与えないことを示す、さまざまな架橋の割合で同様弾力性を持っている。これと?...

静的および動的な基板は、細胞機能に対する組織弾性または剛性の効果を研究するために開発された生体材料の二つのカテゴリーである。静的基板は、それらが製造された後、および/または細胞がプレーティングされれば、それらの物理的特性を変更することはできません。ポリアクリルアミド（PA）ゲルはメカノ調査5,17ために合成した第1の2次元、静的な基質であった。 PAゲルは、調製が容易で、安価で、多用途であり、弾性率​​の広い範囲で製造できる。これらの技術的利点は、PAが一般的に適用される基板をゲル化することが、静的な基質は、細胞外マトリックス（ECM）の動的な性質を示すものではないし、インビボで細胞環境を取り囲む。例えば、ECMは、傷害、開発、または疾患の結果としての剛性の変化を受ける。ダイナミック基板は、したがって、メカノ研究における組織模倣基質モデルとして好まれる<sup&gt; 22,24,25。 多数の合成は、天然の、二次元、三次元の、静的および動的な生体材料は、組織1,3,6,16,23,26剛性を模倣するために開発されている。いくつかの動的な基板は、それらの機械的特性2,4,7,8,12,15,16を変更するために、熱、紫外線、電流、イオン、pH変化が必要ですが、これらの刺激は、ヒドロゲルのバイオアプリケーションを制限することができます。 DNA架橋ポリアクリルアミドヒドロゲル（DNAゲル）は、動的二次元弾性基質である。 DNAの架橋は、メディアへの一本鎖DNA（ssDNA）の添加によってDNAゲルの剛性の時間的、空間的、可逆変調のために許可または9-11,13,14,18,21バッファ 。刺激は弾力性の調節のために適用され、上記の動的なゲルとは異なり、DNAゲルは、弾力性の変化のために適用された一本鎖DNAの拡散に依存している。したがって、細胞を増殖さ上部ゲル表面は、レートoをので、変調された第1の領域であるF弾性変調はゲルの厚さに依存している。 DNAゲルは、しかしながら、ビス-アクリルアミド架橋はDNA（ 図1）からなる架橋で置き換えられ、それらはポリアクリルアミド骨格を有するという点で、それらのPAゲル対応物に類似している。二つのssDNA（SA1とSA2）は、ゲルのDNA架橋を補うために、架橋剤ストランド（L2）とハイブリダイズ。 SA1とSA2は、両方のPAネットワークへの効果的な取り込みのために、5 &#39;端にアクリダイト修正が含まれている明確な配列を有する。ゲル、SA1およびSA2の調製のために個別にPA主鎖に重合されると、その後、重合されたSA1およびSA2を一緒に混合する。 L2、架橋剤は、SA1およびSA2混合物に添加される。 L2の塩基配列は、両方SA1およびSA2配列に相補的であり、L2は、DNA架橋を形成するSA1プラスSA2とハイブリダイズする。初期、DNAゲルの弾性は、L2濃度および架橋（ 表1の両方によって決定される</strong&gt;と2）。 SA1およびSA2は、（100％ゲルと呼ぶ）L2 100質量％架橋されているため、L2、SA1およびSA2の等しい化学量論量を含むDNAゲルは、硬いゲルである。 DNA架橋の低い割合でのL2結果のより低い濃度であり、したがって、よりソフトなDNAゲル。 （50％ゲルと呼ぶ）50％架橋された程度の低いゲルは9-11構築されている。 <img alt="図1" src="/files/ftp_upload/51323/51323fig1highres.jpg" /> 図1：DNAゲル架橋およびuncrosslinking概略図9-11,13,14,18,21手順1：SA1（赤）とSA2（青）を個別にポリアクリルアミドバックボーン（黒）に重合される。重合後、SA1およびSA2重合溶液が一緒に混合される。ステップ2：L2（緑）を添加し、ゲルの架橋を形成するSA1プラスSA2とハイブリダイズされる。ステップ3：R2がtとハイブリダイズ彼は、L2の足掛かり。ステップ4：R2の足掛かりハイブリダイゼーションは、SA1とSA2からL2の解凍を推進。 PAゲルとは異なり、DNAゲルは、合成後に硬くかつ柔らかくすることができます。そのため、DNAのゲル上で増殖した細胞は、動剛性の変化に供することができる。細胞接着性ゲルを補強するために、L2は、架橋の割合を増加させるために低いパーセンテージのゲルの培養培地に添加することができる。細胞接着性ゲルを柔らかくするために、L2は、架橋10,13,21の割合を減少させるために除去することができる。 L2は、L2は、SA1およびSA2（ 表1）からuncrosslinkできるように、3 &#39;端に付加的な足掛かり配列を有する。 L2の除去は、R2と呼ばれる反転鎖のハイブリダイゼーションによって達成される。 R2はL2の全長に相補的であり、L2の足掛かりを最初にハイブリダイズする。足掛かりハイブリダイゼーションは架橋を排除し、ゲル剛性を減少させSA1とSA2からL2の解凍を推進する。 本報告ではとビデオは、段階的な手順は、DNAゲルを補強し、軟化させるの調製のために設けられている。 100％および80％ゲル製剤が記載されているが、このプロトコルは、他の初期および最終の架橋割合のDNAゲルを作成するために調整することができる。一般に、100％および80％ゲルを用意し、ガラスカバースリップ上に固定化、官能化、および細胞を播種。 L2は、80％ゲルの培地に添加され、R2は、100％のゲル、メッキの48時間後の培地に添加する。メディアへのR2の添加が80％架橋、100％ゲルを柔らかくし、一方メディアにL2の添加は、架橋された100％、80％ゲルを硬くする。剛化ゲルを80→100％ゲル軟化ゲルとして指定されているテキストで100→80％のゲルとして指定されている。制御または静的ゲル、ssDNAのためにTsとか​​ら成るかのように、100％および80％ゲルの別のセットに配信される。弾性変調次の二日間の最小限の後、細胞を処理し、分析することができる。 <table border="0" cellpadding="0" cellspacing="0" fo:kEEP-together.within-ページ= &quot;常に&quot;&gt; <tbody align="center"><tr><td rowspan="2"></td><td rowspan="2"> DNAクロスリンク </td><td rowspan="2"> 塩基の数 </td><td> シーケンス（ 足掛かり ） </td><td rowspan="2"> 修飾 </td><td rowspan="2"> 融解温度（T mは 、°C） </td><td rowspan="2"> コメント </td></tr><tr><td> 5 &#39;→3&#39; </td></tr><tr><td rowspan="4"> デザイン1 </td><td> SA1 </td><td> 10 </td><td> GCA CCT TTG C </td><td> 5 &#39;アクリダイト</td><td> 34.9 </td><td></td></tr><tr><td> SA2 </td><td> 10 </td><td> GTC AGA ATG A </td><td> 5 &#39;アクリダイト</td><td> 23.6 </td><td></td></tr><tr><td> L2 </td><td> 30 </td><td> TCA TTC TGA C GC AAA GGT GC G CTA CAC TTG </td><td></td><td> 56 </td><td>10 bpの足掛かりシーケンスが含まれています。 </td></tr><tr><td> R2 </td><td> 30 </td><td> CAA GTGタグCGC ACC TTT GCG TCA GAA TGA </td><td></td><td></td><td> R2はL2に相補的である</td></tr><tr><td rowspan="4"> デザイン2 </td><td> SA1 </td><td> 14 </td><td> CGT GGC ATA GGA CT </td><td> 5 &#39;アクリダイト</td><td> 46.​​9 </td><td></td></tr><tr><td> SA2 </td><td> 14 </td><td> GTT TCC CAA TCA GA </td><td> 5 &#39;アクリダイト</td><td> 40.2 </td><td></td></tr><tr><td> L2 </td><td> 40 </td><td> TCT GAT TGG GAA AC A GTC CTA TGC CAC G GT TAC CTT CAT C </td><td></td><td> 65.9 </td><td> 12 bpの配列の足掛かりは含まれています。 </td></tr><tr><td> R2 </td><td> 40 </td><td> GAT GAA GGT AAC CGT GGC ATA GGA CTG TTT CCC AAT CAG A </td><td></td><td> 65.9 </td><td> R2はL2に相補的である</td></tr><tr><td rowspan="3"> 療nは3 </td><td> SA1 </td><td> 20 </td><td> ACG GAG GTG TAT GCA ATG TC </td><td> 5 &#39;アクリダイト</td><td> 55 </td><td></td></tr><tr><td> SA2 </td><td> 20 </td><td> CAT GCTタグGGA CGA CTG GA </td><td> 5 &#39;アクリダイト</td><td> 56.6 </td><td></td></tr><tr><td> L2 </td><td> 40 </td><td> TCC AGT CGT CCC TAA GCA TG G ACA TTG CAT ACA CCT CCG T </td><td></td><td> 68.8 </td><td>足掛かりは含まれていません。 </td></tr><tr><td rowspan="2"> コントロール </td><td rowspan="2">コントロール</td><td rowspan="2"> 20-40 </td><td> AAA AAA（ など ）や</td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td></tr><tr><td> TTT TTT（ その他 ） </td></tr></tbody></table>セルラーと機械の研究をd、いくつか利用している。のssDNA 9-11,13,14,18,21表1に塩基配列ifferent架橋設計は、静的および動的機械的特性の範囲を有するDNAゲルを生成する。架橋設計において変調パラメータは、塩基配列と、配列の長さまたは架橋の長さである。太字やイタリック体のフォントは、SA1とL2との間にそれぞれSA2とL2の間の塩基対形成を示している。 <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody align="center"><tr><td rowspan="2"></td><td colspan="8"> デザイン </td></tr><tr><td colspan="3"> 1 </td><td colspan="3"> 2 </td><td colspan="2"> 3 </td></tr><tr><td> アクリルアミド濃度（％） </td><td colspan="3"> 10 </td><td colspan="3"> 10 </td><td> 10 </td><td> 4 </td></tr><tr><td> SA1プラスL2にハイブリダイズSA2（％架橋） </td><td> 50 </td><td> 80 </td><td> 100 </td><td> 50 </td><td> 80 </td><td> 100 </td><td> 100 </td><td> 100 </td></tr><tr><td> &lt;強い&gt;弾力性（キロパスカル、平均±SEM） </td><td> 6.6±0.6 </td><td> 17.1±0.8 </td><td> 29.8±2.5 </td><td> 5.85±0.62 </td><td> 12.67±1.33 </td><td> 22.88±2.77 </td><td> 25.2±0.5 </td><td> 10.4±0.6 </td></tr></tbody></table> DNAゲルの表2ヤング率（E）9-11,13,14,18,21。アクリルアミド濃度、架橋の割合、および架橋の長さはDNAゲルに変調することができる。設計1,2、及び3は、それぞれ、20,28、および40 bpの架橋長さを有する。すべてのデザインのための100％のゲルは、ゲル弾性に影響を与えない架橋長さを示す類似の弾性率を持っている。しかし、アクリルアミド濃度のばらつきはDNAゲルの弾性を変化させる。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>ssDNA</td>
			<td>Integrated DNA Technologies (Coralville, Iowa) idtdna.com</td>
			<td></td>
			<td>Do not vortex ssDNA. Gentle invert the vial and/or pipette solution to mix.</td>
		</tr>
		<tr>
			<td>PBS with calcium and magnesium</td>
			<td></td>
			<td></td>
			<td>Any brand.</td>
		</tr>
		<tr>
			<td>100X Tris-EDTA buffer (TE buffer)</td>
			<td>Sigma-Aldrich (St. Loius, MO) sigmaldrich.com</td>
			<td>T9285</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Tris-Borate-EDTA buffer (TBE buffer)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>93290</td>
			<td>TBE is a reproductive toxin.</td>
		</tr>
		<tr>
			<td>40% Acrylamide solution</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>BP14021</td>
			<td>Acrylamide is a toxin.</td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>A3678</td>
			<td>Prepare a 2% solution in TE buffer. APS is a toxin and irratant.</td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>T9281</td>
			<td>Prepare a 20% solution in TE buffer. TEMED is flammable, a corrosive, and a toxin.</td>
		</tr>
		<tr>
			<td rowspan="2">12-mm diameter round coverglass</td>
			<td rowspan="2">Fisher Scientific (Pittsburg, PA) fishersci.com</td>
			<td rowspan="2">12-545-82</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Norland optical adhesive 72</td>
			<td>Norland Products (Cranbury, NJ) norlandprod.com</td>
			<td>NOA72</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">24-well tissue culture plate</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">Any brand.</td>
		</tr>
		<tr>
			<td rowspan="2">Microcentrifuge tubes</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">Any brand.</td>
		</tr>
		<tr>
			<td>Sulfo-SANPAH</td>
			<td>ProteoChem or Thermo Fisher, (Rockland, IL) proteochem.com or thermofisher.com</td>
			<td>C111 or 22589</td>
			<td>Prepare a 0.315 mg/ml solution in water immediately before use. Dissolve at 37&#176;C and filter sterilze. It is normal to observe undisolved sulfo-SANPAH in the filter. Sulfo-SANPAH is light sensitive and, therefore, the solution should be protect from light until UV exposure.</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine (PDL)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>P6407</td>
			<td>Prepare a 0.2 mg/ml solution in water and filter sterilize.</td>
		</tr>
		<tr>
			<td>Collagen Type I</td>
			<td>Affymetrix (Santa Clara, CA) affymetrix.com</td>
			<td>13813</td>
			<td>Prepare a 0.2 mg/ml solution in 0.2 N acetic acid. Solution needs to remain cold at all times to avoid polymerization. Acetic acid is a flammable, toxic, and corrosive.</td>
		</tr>
		<tr>
			<td>22 X 60 cover glass</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>12-544-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive-displacement pipette</td>
			<td>Gilson, Inc (Middletown, WI) gilson.com</td>
			<td>F148504</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>11-718</td>
			<td></td>
		</tr>
		<tr>
			<td>UV light source</td>
			<td></td>
			<td></td>
			<td>Place gels as close as possible to the UV light. UV light can cause skin or eye injury.</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td></td>
			<td></td>
			<td>Any brand.</td>
		</tr>
		<tr>
			<td>Nitrogen gas</td>
			<td>GTS-Welco (Flemington, NJ) www.praxairmidatlantic.com/</td>
			<td>NI 5.0UH-R</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of dna-crosslinked polyacrylamide hydrogels

メカノは、細胞の形態と機能を向ける内の物理的な手がかりの重要な役割に対処する新たな科学的な領域です。組織の剛性は、疾患、発達、および傷害に調節するため、例えば、細胞機能に対する組織の弾性の影響は、メカノ研究の主要な領域である。細胞がプレーティングされると剛性を変えることができない静的な組織模倣物質、または物質は、主に細胞の機能上の組織の剛性の影響を調査するために使用される。静的試験から収集した情報は貴重であるが、これらの研究は、 生体内での細胞の微小環境の動的な性質を示すものではありません。より良い細胞機能に対する動的剛性の影響に対処するために、DNA架橋ポリアクリルアミドヒドロゲルシステム（DNAゲル）を開発した。他の動的基質とは異なり、DNAゲルは、減少または刺激することなく、製造後に剛性が増加する能力を持っている。 DNAゲルは、DNAのcrosslから成りポリアクリルアミドバックボーンに重合されるインク。追加および一本鎖DNAの送達を介して架橋を除去することは、時間、空間、およびゲル弾性の可逆的制御を可能にする。私たちは、DNAゲルの弾力性の動的調節が線維芽細胞と神経細胞の挙動に影響を与えることがこれまでの報告に示されている。本報告書やビデオでは、準備のDNAゲル上でDNAゲルの架橋メカニズムと、ステップ·バイ·ステップの手順を説明する模式図を提供する。

本発明者らの研究に先立って、細胞 - ECM相互作用は、静的に準拠生体材料上または不可逆一方向動的基板上に観察された。これらの基板は、正確な細胞の微小環境の動的な性質を反映していない。私たちの仕事は、動的な生体材料を軟化し、補強に細胞-ECM相互作用を研究するための、より生理的なモデルを提供することで、既存の技術上のパラダイムをシフトする。以前の研究では、これ?...

Watch this Scientific Journal Video about Preparation of DNA-crosslinked Polyacrylamide Hydrogels at JoVE.com

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

当研究室では、より良い細胞機能に組織の硬さを調節する効果を理解するためにDNA架橋ポリアクリルアミドハイドロゲル、動的なヒドロゲル系を開発しました。ここでは、これらのヒドロゲルを調製するための回路図、説明、およびプロトコルを提供する。

DNA架橋ポリアクリルアミドハイドロゲルの調製

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

preparation-of-dna-crosslinked-polyacrylamide-hydrogels

Research

JoVE Journal

Biology

14.4K ビュー.  JFK Medical Center. 当研究室では、より良い細胞機能に組織の硬さを調節する効果を理解するためにDNA架橋ポリアクリルアミドハイドロゲル、動的なヒドロゲル系を開発しました。ここでは、これらのヒドロゲルを調製するための回路図、説明、およびプロトコルを提供する。 

ビデオ: Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

ポリアクリルアミドゲルでのタンパク質の迅速かつ高感度コロイドクマG - 250染色

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

生物学

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

変性尿素ポリアクリルアミドゲル電気泳動（尿素PAGE）

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

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 ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

細胞溶解物からの多タンパク質複合体の解析のための青いネイティブポリアクリルアミドゲル電気泳動（BN - PAGE）

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

軟骨アガロースゲルの機械的刺激

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

三次元ハイドロゲルでイメージングをFRET

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

ポルシン卵母細胞カプセル化のためのタンパク質分解アルギン酸ヒドロゲルおよび疎水性マイクロバイオリアクター

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

ポリアクリルアミドゲル電気泳動と銀染色によるグリコサミノグリカンの検出

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

根の滲出液中の根茎細菌の化学誘引物質の迅速な同定のための改良された気化症アッセイ

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

CFPS反応の各種ハイドロゲルマトリックスへの埋め込み

Methods for Embedding Cell-Free Protein Synthesis Reactions in Macro-Scale Hydrogels

Synthetic gene networks provide a platform for scientists and engineers to design and build novel systems with functionality encoded at a genetic level. While the dominant paradigm for the deployment of gene networks is within a cellular chassis, synthetic gene networks may also be deployed in cell-free environments. Promising applications of cell-free gene networks include biosensors, as these devices have been demonstrated against biotic (Ebola, Zika, and SARS-CoV-2 viruses) and abiotic (heavy metals, sulfides, pesticides, and other organic contaminants) targets. Cell-free systems are typically deployed in liquid form within a reaction vessel. Being able to embed such reactions in a physical matrix, however, may facilitate their broader application in a wider set of environments. To this end, methods for embedding cell-free protein synthesis (CFPS) reactions in a variety of hydrogel matrices have been developed. One of the key properties of hydrogels conducive to this work is the high-water reconstitution capacity of hydrogel materials. Additionally, hydrogels possess physical and chemical characteristics that are functionally beneficial. Hydrogels can be freeze-dried for storage and rehydrated for use later. Two step-by-step protocols for the inclusion and assay of CFPS reactions in hydrogels are presented. First, a CFPS system can be incorporated into a hydrogel via rehydration with a cell lysate. The system within the hydrogel can then be induced or expressed constitutively for complete protein expression through the hydrogel. Second, cell lysate can be introduced to a hydrogel at the point of polymerization, and the entire system can be freeze-dried and rehydrated at a later point with an aqueous solution containing the inducer for the expression system encoded within the hydrogel. These methods have the potential to allow for cell-free gene networks that confer sensory capabilities to hydrogel materials, with the potential for deployment beyond the laboratory.

著者スポットライト:ハイドロゲルに無細胞タンパク質合成反応を埋め込むための新しいアプローチ 

Here, we present two protocols for embedding cell-free protein synthesis reactions in macro-scale hydrogel matrices without the need for an external liquid phase.

マクロスケールハイドロゲルへの無細胞タンパク質合成反応の埋め込み法

Synthetic gene networks provide a platform for scientists and engineers to design and build novel systems with functionality encoded at a genetic level. ...