この研究は、オーストラリア研究評議会(ARC)ディスカバリープログラム(DP150101065)と、低レベル(IDEAL)(IH150100028)でのエンドユーザ分析のための統合デバイスのためのARC研究ハブによって支援されました。

1. tBLMS電極の準備
<ol><li>初の単層コーティングの準備<ol>
<li>ベンジルジスルフィド-テトラ-エチレングリコール-OH「スペーサー」分子の3 mM 1:9比で構成されるエタノール溶液に、スパッタ付きの金パターン電極顕微鏡スライドを浸す(ベンジルジスルフィドは4つの酸素エチレングリコールスペーサーを含み、 OH基)およびベンジルジスルフィド(テトラエチレングリコール)n=2C20-フィタニル「つながれた」分子で終了する。これにより、二重層を固定できる最初の層コーティングが作成されます。 注:金の電極はカスタム25 mm x 75 mmポリカーボネートスライド20に100 nm、99.9995%の金(5n5ゴールド)フィルムを蒸発させることによって作られる。</li>
		<li>室温で第1層を用いて電極を少なくとも1時間インキュベートする。</li>
		<li>30s以上の純エタノールを大量に浸漬して金電極をすすいだ。</li>
		<li>次のステップに直接最初の単層と金電極スライドを使用するか、純粋なエタノールの完全な瓶に保存します。</li>
		<li>注:最初のレイヤーの整合性を確保するには、スライドの金色部分への直接接触を最小限に抑えます。</li>
	</ol></li>
	<li>最初の単層コーティングスライドを組み立てる<ol>
<li>ピンセットを使用して容器から1つのコプラナー金電極スライドを慎重に取り外し、tBLMが形成されるパターン化された領域と接触しないようにしてください。 注:金が堆積しているスライドの側面を識別することに注意してください。</li>
		<li>空気乾燥スライドは1〜2分間、残留エタノールを除去する。</li>
		<li>乾燥した表面の上に金の電極を置き、金の電極が正しく上向きのパターン化された金の表面と向きであることを確認します。</li>
		<li>薄いラミネートから透明な接着剤層カバーを剥がし、6つのチャネルの上に置き、各ウェルを定義します。</li>
		<li>図1Aに示すように、圧力ローラを使用してスライドと透明接着層の間の空気を放出します。 注: このステップに必要な時間は、研究者が最適化する必要があります。このプロトコルでは、時間は2〜3分の範囲です。</li>
		<li>第1層を損傷しないように、第2の脂質二重層を組み立てられた第1単層被覆電極に早急に導入する。</li>
	</ol></li>
	<li>第2脂質二重層の調製<ol>
<li>6つのウェルスライドの最初のウェルに関心のある3 mM脂質の6 μLを加えます。マイクロピペットの先端の端が金の表面に触れないようにしてください。 注:この研究で使用される脂質混合物は、3 mM 70% ジウテリリアニック C20 ジフィタニル-エテル-グリセロホスファチジルコリン (DPEPC) と 30% C20 ジフィタニルジグリセリド エーテル脂質 (GDPE) 3 mM コレステロール-PEG-ビオチンと混合した 50:1 モル比で構成されています。</li>
		<li>脂質混合物の6 μLを各添加の間に10 sギャップを持つ他のウェルに導入する。</li>
		<li>PBSなどのバッファーで電極上で脂質混合物を交換する前に、室温で正確に2分間、各ウェルをインキュベートします。各ウェルが各々2分間脂質でインキュベートされるように、10s離れた添加及び緩衝交換のための時間を空間化する。</li>
		<li>PBSバッファー(pH 7.0)を50 μLで3回洗浄します。電極の上に50 μLのバッファを常に残してください。電極を乾燥させないでください。 注:このように水溶液とエタノール溶媒を置き換える(溶媒交換法)は、つながれた化学を介して金電極に固定された単一の脂質二重層の迅速な形成を可能にする。</li>
	</ol></li>
	<li>電気インピーダンス分光法(EIS)測定を用いたtBLM形成試験<ol>
<li>準備した電極スライドをACインピーダンス分光計(例えばテタポッド)に挿入します。分光計がUSBポートを介してソフトウェアを実行しているコンピュータに接続されていることを確認します。</li>
		<li>ソフトウェアを開き、[セットアップ] をクリックして、[ハードウェア] を開きます。</li>
		<li>25 mV のピークからピークへの交流励起を使用するようにハードウェア設定を設定します。</li>
		<li>0.1~10,000 Hzの周波数を設定し、10年あたり2ステップで高速インピーダンス対策を行いOKを押します。</li>
		<li>[設定] メニューをクリックし、[モデル]を開きます。</li>
		<li>テザリング金電極を直列の定相素子として記述する等価回路モデルを用いて、電解質バッファーを記述する抵抗と、脂質二重層を記述する並列抵抗キャパシタネットワークを有する、OKを押す。</li>
		<li>[開始]ボタンを押して、膜容量(Cm)と膜伝導(Gm)のリアルタイム測定を開始します。典型的なtBLMsのCm値は、10%のテザーケリー21,22に対して12.5 nF~15.5 nFの範囲内にあるべきである。</li>
		<li>プロトコルを実行して実験を終えた後、データを保存します。</li>
		<li>次のウェルで測定を繰り返します。</li>
	</ol></li>
</ol>2.レーザー照射
<ol><li>実験用セットアップ メモ: カスタムメイドのシステムは、各tBLMに対して個別に設定されています。<ol>
<li>レーザーを危険にさらすように防光ボックスで実験を行います。</li>
		<li>光学表を使用して、不要な振動を減らすために実験を設定します。</li>
		<li>金のスライドが接続されているインピーダンスリーダーをXYZステージに置き、レーザー光源の経路に座るような高さを上げます。</li>
		<li>レーザー光源の高さを制御して適切な精度を達成するために、粗い細かい焦点を合わせる顕微鏡ギアリングを使用します。</li>
		<li>電極スライドの縦軸に沿ってレーザーパスをターゲットにします。 注意:常に適切なレーザー安全メガネを着用し、良好なレーザー安全プロトコルを維持します。</li>
		<li>実験を開始する前に、選択した調整されたレーザーを安定させます。 注: 実験用セットアップの概略を図2Aに示します。</li>
	</ol>
</li>
	<li>レーザー電極と金電極のアライメント 注: 始める前に、パワーメーターを使用してレーザー出力を評価し、非常に低いワット数のみがtBLに送られるようにしてください。<ol>
<li>レーザーパスまたは電極の角度を調整して、レーザーが電極を覆う液体を通過し、金の表面で均等に見えるようにします。</li>
		<li>膜伝導の変化を観察しながら、微調整を使用してレーザービーム光源を上げ下げすることにより、実験ごとにレーザー光の位置を調整します。</li>
		<li>導通変化が見られない場合は、ノブをロックしてレーザーパスの位置を固定します。 注: レーザーが下層の金電極と相互作用すると、膜コンダクタンス値が増加します。したがって、そのような相互作用が不可能になるようにレーザー経路を調整することが重要である。</li>
	</ol>
</li>
	<li>サンプル準備<ol>
<li>図2、位置3に示すように、レーザー光の位置合わせ(膜伝導に変化がない場合)を準備します。</li>
		<li>レーザーがOFFの間に tBL が浸漬される PBS バッファに目的の GNP(機能化または裸)を追加します。</li>
		<li>電極に触れないように、tBLMを取り巻くPBSバッファーを3回そっと混ぜます。</li>
		<li>室温で5〜10分間インキュベートします。</li>
		<li>レーザーをオンにしてサンプルを照射し、図2、位置3に見られるように正しい位置のレーザー光の位置を使用します。</li>
		<li>レーザー光波長を使用して、GnMPのサイズ、形状、濃度の適切な組み合わせを使用してください。 注: 設定された波長のレーザービームは、対応するGNPプラズモン共鳴周波数に結合する必要があります。</li>
		<li>連続的に測定された電流を記録する(リアルタイム測定)。</li>
		<li>ステップ 2.2.1 ~ 2.3.7 を実行し、制御実験用の GNP の追加を省略します。</li>
	</ol>
</li>
</ol>3.統計データ分析と表示
<ol><li>データをスプレッドシートにエクスポートします。</li>
	<li>膜伝導パラメータ対時間を抽出します。</li>
	<li>正しい位置でレーザー光を設定した後、およびGnPS導入前に記録されたデータを使用してください。</li>
	<li>基準膜コンダクタンス上で測定された膜伝導率を分割してデータを正規化します。 注:これは導入された照射されたGNPによって引き出される膜伝導値の相対的な変化を確認する。</li>
	<li>時間のプロット(x軸)と正規化された膜伝導(y軸)のデータを提示します。</li>
</ol>4.照射されたナノ粒子からtBLMで発生する局所熱量を予測する(熱予測モデル)
<ol><li>照射されたナノ粒子溶液中の吸収放射線パワーを計算するために、ドンブロフスキー23に従って放射線移動の問題を解決する。</li>
	<li>吸収された放射による熱源をエネルギー方程式に組み込むことにより、発熱を計算します。 注: 照射されたナノ粒子とナノ材料電極界面からのtBLMにおける発熱の数値解析の詳細については、19を参照してください。</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle-mediated+cellular+response+is+size-dependent.">Nanoparticle-mediated cellular response is size-dependent.</a> Nature Nanotechnology. 3 (3), 145 (2008).</li><li>He, C., Hu, Y., Yin, L., Tang, C., Yin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+particle+size+and+surface+charge+on+cellular+uptake+and+biodistribution+of+polymeric+nanoparticles.">Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles.</a> Biomaterials. 31 (13), 3657-3666 (2010).</li><li>Wang, F. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+and+bulk+contributions+to+the+second-order+nonlinear+optical+response+of+a+gold+film.">Surface and bulk contributions to the second-order nonlinear optical response of a gold film.</a> Physical Review B. 80 (23), 233402 (2009).</li></ol>

著者らは、潜在的な競合する利益とみなされる可能性のある次の財政的利益/個人的な関係を宣言する:ブルース・コーネル教授は、外科診断SDxテザリングメンブレンPty株式会社の科学技術ディレクターである。

このプロトコルは、金ナノ粒子のレーザー照射に応答してリアルタイムの電気インピーダンス記録を可能にする水平レーザーアライメントセットアップと組み合わせて、同一平面電極基板を備えたtBLMモデルの使用を記述する。ここで提示されるEIS記録の方法は、膜全体のイオン電流変化の記録を提供するために必要な実験の最小限のリストを構築し、これは、結合レ...

照射された金ナノ材料の高熱性能は、感染症および腫瘍に対する低侵襲、選択的、標的治療の新しいクラスを提供する。レーザーで加熱できるナノ粒子の雇用は、疾患細胞を選択的に破壊するとともに、選択的薬物送達2,3の手段を提供するために用いられてきた。熱いプラズモニックナノ粒子の光熱による現象の結果は、細胞膜への損傷である。この流体脂質二重層膜は、細胞膜全体のイオンポテンシャル勾配を維持するために多くのタンパク質が存在するため、固有の膜タンパク質の変性および膜損傷も細胞死に至る可能性があるため、このような治療を受けている細胞にとって特に脆弱な部位と考えられている。ナノスケールでの熱伝達を決定および監視する能力は、照射されたGnP1、5、6、7、GnPsとバイオ膜間の分子相互作用の評価と理解、ならびに生物学的組織における組み込みGnMPのレーザー誘発加熱現象の直接的な結果の研究と応用に重要な関心事であるが、 まだ完全に解明されていない8.したがって、照射されたGNPの温熱法プロセスを十分に理解することは依然として課題である。このように、細胞の自然環境を模倣するナノ材料電極界面の開発は、生物学的システム内で照射された金ナノ粒子の熱伝達特性の詳細な調査を行う手段を提供することができる。
ネイティブ細胞膜の複雑さは、細胞内の照射されたGNP相互作用を理解する上で重要な課題の1つです。天然脂質膜アーキテクチャおよび機能性の近接的なバイオ模倣バージョンを提供するために開発された様々な人工膜プラットフォームが存在するが、これらに限定されないが、黒色脂質膜9、支持された平坦二重層膜10、ハイブリッド二層膜11、ポリマークッション脂質二層膜12およびテザリング二層脂質膜13。各人工脂質膜モデルは、天然脂質膜14を模倣することに関して明確な利点および限界を有する。
本研究では、tBLMモデルを用いて、金ナノ粒子と脂質膜相互作用を評価するためのセンサーとしての脂質膜被覆電極の採用について説明する。tBLMベースのバイオセンサ検出スキームは、わずか量の膜損傷が15、16、17、18に至る他のシステム(パッチクランプまたはリポソームによって形成された膜など)とは異なり、テザード膜が自己修復できる固有の安定性と感度13を提供する。また、tBLMはmm2次元であるため、背景インピーダンスはパッチクランプ記録技術よりも桁違いに低く、ナノ粒子相互作用による基底膜イオン束の変化を記録することができます。この結果、本プロトコルは、135 nW/μm2と低いパワーを持つレーザーによって励起される結合されたGnMPによる膜伝導度の変化を対比することができる。
ここで提示されるシステムは、正確なレーザーパラメータ、粒子サイズ、粒子コーティング、および熱療法の設計と開発に必要な組成を決定するための敏感で再現可能な方法を提供します。これは、新たな光熱療法の改良に不可欠であり、生物学的システム内の熱伝達の詳細なメカニズムに関する貴重な情報を提供する。提示されたプロトコルは、以前に公開された作品19に基づいています。プロトコルの概要は次のとおりです: 最初のセクションは、tBLM の形成を定義します。2 番目のセクションでは、セットアップを構築し、励振レーザー ソースを位置合わせする方法について説明します。最後のセクションでは、電気インピーダンス分光法データから情報を抽出する方法を示しています。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30 nm diameter streptavidin-conjugated gold nanoparticles</td>
			<td>Cytodiagnostics</td>
			<td>AC-30-04-05</td>
			<td>This is a streptavidin-conjugated GNPs product ready for use</td>
		</tr>
		<tr>
			<td>30 nm diameter bare gold nanoparticles</td>
			<td>Sigma-Aldrich</td>
			<td>753629</td>
			<td>This is a bare GNPs product ready for use</td>
		</tr>
		<tr>
			<td>Cholesterol-PEG-Biotin (MW1000)</td>
			<td>NANOCS</td>
			<td>PG2-BNCS-10k</td>
			<td>Dissolved in highly pure ethanol</td>
		</tr>
		<tr>
			<td>C20 Diphytanyl-Glycero-Phosphatidylcholine lipids</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-S1</td>
			<td>1 ml glass vial containing 70% C16 diphytanyl phosphatidylcholine (DPEPC) and 30% C16 diphytanyl glycerol (GDPE) in 99.9% ethanol</td>
		</tr>
		<tr>
			<td>Benzyl-disulfide-tetra-ethyleneglycol-OH</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-S2</td>
			<td>Spacer molecules</td>
		</tr>
		<tr>
			<td>Benzyl-disulfide (tetra-ethyleneglycol) n=2 C20-phytanyl&#160;</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-S2</td>
			<td>Tethered molecules</td>
		</tr>
		<tr>
			<td>532 nm green laser continuous light</td>
			<td>OBIS LS/OBIS CORE LS, China</td>
			<td>ND-1000</td>
			<td>The power of this laser was ~135&#160;mW&#160;</td>
		</tr>
		<tr>
			<td>tethaPod EIS reader</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-R1</td>
			<td>A reader of conductance and capacitance on six channels simultaneously</td>
		</tr>
		<tr>
			<td>tethaPlate cartridge assembly</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-BG</td>
			<td>Materials to attach the slide with electrodes to the flow cell cartridge</td>
		</tr>
		<tr>
			<td>Clamp and slide assembly jig</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-A1</td>
			<td>Materials to attach the slide with electrodes to the flow cell cartridge</td>
		</tr>
		<tr>
			<td>Lipid coated coplanar gold electrodes</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-T10</td>
			<td>Coplanar&#160; gold electrodes are made from 25 mm x 75&#160;mm x&#160;1&#160;mm polycarbonate base substrate with patterned gold arrays layout, then coated with benzyldisulphide, bis-tetraethylene glycol C16 phytanyl half membrane spanning tethers in a tether ratio of 10%&#160;</td>
		</tr>
		<tr>
			<td>tethaQuick software</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-B1</td>
			<td>Software for use with tethaPod to process data and display conductance, impedance and capacitance measurements from the tethaPlate electrodes</td>
		</tr>
		<tr>
			<td>&#160;99.9% Pure ethanol</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>34963</td>
			<td>Absolute,&#160; 99.9%</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td>pH 7</td>
		</tr>
	</tbody>
</table>

tethered bilayer lipid membranes to monitor heat transfer between gold nanoparticles and lipid membranes

ここでは、金電極に組み立てられたテザリング二重層脂質膜(tBLM)を用いた電気化学による、照射された金ナノ粒子(GNP)と二層脂質膜の間の熱伝達を調べるプロトコルを報告する。ストレプトアビジン共役GNPなどの照射された修飾GNPは、ビオチンなどの標的分子を含むtBLMに埋め込まれています。このアプローチを用いることで、照射されたGNPと目的のエンティティを持つモデル二重層脂質膜との間の熱伝達プロセスは、水平に焦点を合わせるレーザービームによって媒介される。熱予測計算モデルは、tBLMにおける電気化学的に誘導された伝導度の変化を確認するために使用されます。使用される特定の条件下では、熱パルスを検出するには金ナノ粒子を膜表面に特定の付着させる必要がありますが、結合されていない金ナノ粒子は測定可能な応答を引き出すことができませんでした。この技術は、レーザーパラメータ、粒子サイズ、粒子コーティングおよび組成物の最適化を可能にする熱療法の戦略の設計と開発に直接利用することができる強力な検出バイオセンサーとして機能します。

tBLM を作成できるゴールド基板を図 1に示します。実験用セットアップの概略を図2に示します。
図1Aに示すように、コプラナー金電極は、パターン化された金アレイを備えた25 mm x 75 mm x 1 mm ポリカーボネートベース基板から作られています。透明な接着剤層は6つの個々の測定室を定義する。同一平面の金...

Watch this Scientific Journal Video about Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes at JoVE.com

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

この研究は、レーザー照射金ナノ粒子からtBLMへの熱伝達の動的で非侵襲的なモニタリングを達成するためのプロトコルを概説する。このシステムは、インピーダンス分光法を組み合わせて、tBLM全体の伝導変化をリアルタイムで測定し、金ナノ粒子照明を駆動する水平に焦点を当てたレーザービームを熱製造に使用します。

金ナノ粒子と脂質膜の熱伝達をモニターする二重層脂質膜

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

tethered-bilayer-lipid-membranes-to-monitor-heat-transfer-between

Research

JoVE Journal

Biology

2.6K ビュー.  University of Technology Sydney. この研究は、レーザー照射金ナノ粒子からtBLMへの熱伝達の動的で非侵襲的なモニタリングを達成するためのプロトコルを概説する。このシステムは、インピーダンス分光法を組み合わせて、tBLM全体の伝導変化をリアルタイムで測定し、金ナノ粒子照明を駆動する水平に焦点を当てたレーザービームを熱製造に使用します。

ビデオ: Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Method for the Isolation of Francisella tularensis Outer Membranes

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other bacterial pathogens, the extremely low infectious dose (&lt;10 CFU), rapid disease progression, and high morbidity and mortality rates suggest that the virulent strains of Francisella encode for novel virulence factors. Surface-exposed molecules, namely outer membrane proteins (OMPs), have been shown to promote bacterial host cell binding, entry, intracellular survival, virulence and immune evasion. The relevance for studying OMPs is further underscored by the fact that they can serve as protective vaccines against a number of bacterial diseases. Whereas OMPs can be extracted from gram-negative bacteria through bulk membrane extraction techniques, including sonication of cells followed by centrifugation and/or detergent extraction, these preparations are often contaminated with periplasmic and/or cytoplasmic (inner) membrane (IM) contaminants. For years, the "gold standard" method for the biochemical and biophysical separation of gram-negative IM and outer membranes (OM) has been to subject bacteria to spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation. Once layered on a sucrose gradient, OMs can be separated from IMs based on the differences in buoyant densities, believed to be predicated largely on the presence of lipopolysaccharide (LPS) in the OM. Here, we describe a rigorous and optimized method to extract, enrich, and isolate F. tularensis outer membranes and their associated OMPs.

Method for the Isolation of Francisella tularensis Outer Membranes

A protocol for separating inner and outer membranes from Francisella tularensis by spheroplasting, osmotic lysis, and sucrose density gradient ultracentrifugation.

の分離のための方法野兎病菌外膜

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other ...

生物学

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

グラミシジンベースの蛍光アッセイ、脂質二重層のプロパティの変更の可能性小分子を決定するための

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

回路基板と生体膜のモデル化と軸索の電気信号の測定：学生研究室の演習を

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

磁気活性化細胞選別（LIMACS）を使用して、脂質小胞を介したアフィニティクロマトグラフィー：タンパク質脂質相互作用を解析する新手法

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

構造と機能研究のための脂質膜へのKvチャネルの再構築

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

腸によって脂質輸送を研究するためのCaco-2細胞を用いました

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.

Lipid polyesters constitute the structural components of two cell wall modifications, the plant cuticle and suberin-containing diffusion barriers. In this video, we describe a method to depolymerize cutin from whole delipidated leaves. The method can be applied to investigating mutants compromised in either cutin or suberin biosynthesis.

植物キューティクル脂質ポリエステルモノマーの単離と組成分析

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

脂質豊かさとナイルの赤とオイル赤 O 染色により線虫の脂質分布評価の定量化

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

細胞外のフラックスを用いた Explanted 網膜組織のエネルギー代謝の測定

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

ショウジョウバエ幼虫における大鼻細胞の解剖と脂質液滴染色

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...