この作品は、スロベニアの研究機関（プロジェクトJ2 - 9764とプログラムP2 - 0249）によってサポートされていました。このビデオでは、&quot;エレクトロポレーションベースの技術と治療&quot;リュブリャナ、スロベニアの大学で電気工学の学部が主催する科学的なワークショップや大学院のコースのための補助教材を表します。

I.はセルトラッカーCMFDAとCMRAで細胞をロードする<ol><li>実験は、マウスのメラノーマ細胞株（B16 - F1）の事前に準備された細胞について実施した。細胞は、投与16、0.15 mg / mlのL -グルタミン、10％ウシ胎児血清を含むDMEM培地（ダルベッコ改変イーグル培地）で70〜80％コンフルエントになる2つの分離25cm 2の培養フラスコ（TPP、ZDA）で栽培されています/ mlのゲンタマイシン（Sigma - Aldrich社、ドイツからのすべて）、200単位/ ml crystacillin（Pliva、クロアチア）、および37℃、5％CO 2でインキュベート℃の</li><li>オリジナルの染料の50μgのために（Sigma - Aldrich社、ドイツ）のDMSO（グリーンCMFDA用とオレンジCMRAため、それぞれ）10.76μlを、9μlを添加して細胞のトラッカー（インビトロジェン社、米国）の2つの10 mMのストック溶液を準備するインビトロジェンのバイアル。ストック溶液は、℃で数ヶ月間で4冷蔵庫で保存することができます。 DMSOの結晶が溶解するまで実験を開始する前に、ソリューションのウォームアップを行います。 </li><li>重炭酸フリークレブス- Hepes緩衝液（130 mMのNaCl、4.7 mMの塩化カリウム、1.2 mMのMgSO 4を 、1.2 mMのKH 2 PO 4、29.6 mMのD -グルコース、1.3 mMのCaCl 2を 、10mMのHEPES、pH7.4）を準備する。 2つの15エッペンドルフチュー​​ブに別々に重炭酸フリークレブス- Hepes緩衝液3mlに各ストック溶液2.1μL（それぞれ10 mMのCMFDAまたはCMRAを、）ミックス。これは約7μMCMFDAを（またはCMRA）を含む&quot;ローディングソリューション&quot;を得られます。 </li><li>重炭酸フリークレブス- HEPES緩衝液で細胞を2回リンスし、フラスコにソリューションを読み込んで挿入。 37℃5％CO 2で30分間細胞をインキュベート℃にこの最初のインキュベーションの試薬は、細胞膜を自由に通過する時には、いったん細胞の中に、試薬は、細胞非透過性の蛍光反応生成物に変換されます。 </li><li> 37で最初のインキュベーションの後、リンス、5％CO 2でさらに2時間培養培地で細胞をインキュベート℃に</li><li>両方のフラスコに細胞をトリプシン処理（CMFDAとCMRA搭載）および50 ml遠心チューブに比1:1（TPP、ZDA）で一緒に赤と緑のセルを混在させること。 DMEM培地で希釈することによりまたは遠心分離による濃縮により、5 × 10 6細胞/ mlに細胞濃度を調整します。 24マルチウェルプレート（TPP、ZDA）の各ウェルに細胞懸濁液の20μlのドロップを置きます。 ℃で20分間、ややもの表面に付着し、細胞の接触を確立できるように37℃、5％CO 2で細胞をインキュベートする。 </li></ol> II。電気融合<ol><li> isoosomolarリン酸カリウム緩衝液（10mMのKH 2 PO 4、10mMのK 2 HPO 4、1mMのMgCl 2を 、250mMスクロース）とhypoosmolarリン酸カリウムバッファー（10 mM KH 2 PO 4、10mMのK 2 HPO 4、1mMのMgCl 2を 、75 mMスクロース）の準備。 </li><li> 、顕微鏡ステージ上に細胞をマルチウェルを配置ウェルの下部に位置電極を、パルス発生器に接続します。 </li><li> isoosomolarリン酸カリウム緩衝液1mlで細胞を洗浄。細胞の腫脹を誘発するためにhypoosmolarリン酸カリウム緩衝液350μlを添加します。バッファは、電極をカバーする必要があります。 </li><li>電気パルスを適用する前に2分間hypoosmolarバッファーで細胞を残す。この時間の間にインテリアや細胞の外側との間の浸透圧の不均衡に起因する細胞内への水分の流入は、細胞容積の増加を引き起こす。細胞は彼らの最大のボリュームに近づく際には、電気パルスが調節容量の減少を開始する前に、適用する必要があります。 </li><li>最適な電気融合を実現し、細胞の生存を維持するために、電気パルスの最適なパラメータを使用する必要があります。これらは、[1]を使用する細胞株に依存する。この実験では、8の矩形パルス（1 Hzで100μsの間の持続時間と各）の列車は、エレクトロポレーションのデバイスを（このケースCliniporator、ジェーア、イタリア）を使用して、各サンプルに適用されます。パルスは、よく制御を除いて、各ウェルに電極間に約1200 V / cmの電場を作る、0.8mmの直径とそれらの間に5 mmの距離で二つの平行な白金/イリジウムワイヤー電極に配信されます。 </li><li>パルスの配達後10分間静細胞を残す。蛍光および位相差顕微鏡による核融合の利回りを決定します。 </li></ol> III。画像取り込みと融合の収量の決定<ol><li>細胞は、目的（x20）をと冷却CCDカメラ（VisiCam 1280、Visitron、ドイツ）を装備した蛍光顕微鏡を（我々の場合ツァイスAxioVert 200、ツァイス、ドイツ）を用いて観察される。画像は、MetaMorph 7.1.1（Molecular Devices社、米国）に買収されていますが、他の同じような買収のソフトウェアを使用することもできます。 CMFDAは548 nmで492 nmの単色光分光器（ポリクロームIV、Visitron、ドイツ）とCMRAで興奮している。 CMFDAとCMRAの蛍光は（D605/55m、Cの2つの発光535 nmの中心フィルター、つ（HQ535/30m、CMFDA用）と510nmで他の中心を使用して取得されるMRA、クロマ、米国）の両方。ダイクロイックミラー（Q515LP）を使用すると、チャンネルのクロストークを防止。 </li><li>各ウェルに5つのランダムに選択されたフィールドには3つのイメージを（位相コントラスト、赤色および緑色蛍光）を取得。各画像の三つ組から3チャンネルの画像を作成します。そのようなイメージでは蛍光を発する細胞質は細胞膜と一緒に見ることができます。融合細胞は、このように容易に決定することができます[図1]。 </li><li>各3チャンネルの画像で細胞（赤、緑、二重に蛍光）の3種類すべてをカウントします。各画像のすべてのセルの数を二重に蛍光細胞の数を割ることによって二重に蛍光細胞の割合を決定する。核融合の利回りは、融合細胞の半分から2を乗じて二重に蛍光細胞が（同じ色のヒューズの際の細胞）が検出されていないのパーセンテージとして定義されています。 </li></ol>代表的な結果<img src="/files/ftp_upload/1991/1991fig1.jpg" alt="図1" /> 図1電気融合後のB16F1細胞の3チャネルの顕微鏡像：。位相コントラスト、CMRA蛍光（548 nmで励起）とCMFDA蛍光（492 nmで励起）、客観的な倍率20倍

<ol>
	<li>&#268;emazar, M., Jarm, T., Miklav&#269;i&#269;, D., Ma&#269;ek-Lebar, A., Ihan, A., Kopitar, N. A., Ser&#353;a, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+electric-field+intensity+on+electropermeabilization+and+electrosensitivity+of+various+tumor-cell+lines+in+vitro.">Effect of electric-field intensity on electropermeabilization and electrosensitivity of various tumor-cell lines in vitro.</a> Electro. Magnetobiol. 17, 263-272 (1998).</li><li>Trontelj, K., Reber&#353;ek, M., Kandu&#353;er, M., &#269;urin &#353;erbec, V., Miklav&#269;i&#269;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+bulk+cell+electrofusion+in+vitro+for+production+of+human-mouse+heterohybridoma+cells.">Optimization of bulk cell electrofusion in vitro for production of human-mouse heterohybridoma cells.</a> Bioelectrochemistry. 74, 124-129 (2008).</li><li>Rols, M. -. P., Teissi&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+electrically+induced+permeabilization+and+fusion+of+Chinese+hamster+ovary+cells+by+osmotic+pressure.">Modulation of electrically induced permeabilization and fusion of Chinese hamster ovary cells by osmotic pressure.</a> Biochemistry. 29, 4561-4567 (1990).</li><li>Neil, G., Zimmermann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrofusion.">Electrofusion.</a> Methods in Enzymology. 220, 174-196 .</li><li>Abidor, I. G., Li, L. -. H., Hui, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+cell+pellets:+II.+Osmotic+properties,+electroporation,+and+related+phenomena:+Membrane+interactions.">Studies of cell pellets: II. Osmotic properties, electroporation, and related phenomena: Membrane interactions.</a> Biophysical journal. 67, 427-435 (1994).</li><li>Jaroszeski, M. J., Gilbert, R., Fallon, P. G., Heller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanically+facilitated+cell-cell+electrofusion.">Mechanically facilitated cell-cell electrofusion.</a> Biophys J. 67 (4), 1574-1581 (1994).</li><li>Ramos, C., Bonefant, D., Teissi&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+hybridization+by+electrofusion+on+filters.">Cell hybridization by electrofusion on filters.</a> Analytical biochemistry. 302, 213-219 (2002).</li><li>Cao, Y., Yang, J., Yin, Z. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+high-throughput+cell+electrofusion+in+a+microelectrode-array+chip.">Study of high-throughput cell electrofusion in a microelectrode-array chip.</a> Microfluid Nanofluid. 5, 669-675 (2008).</li><li>U&#353;aj, M., Trontelj, M., Kandu&#353;er, M., Miklav&#269;i&#269;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+size+dynamics+and+viability+of+cells+exposed+to+hypotonic+treatment+and+electroporation+for+electrofusion+optimization.">Cell size dynamics and viability of cells exposed to hypotonic treatment and electroporation for electrofusion optimization.</a> Radiol. Oncol. 43, 108-119 (2009).</li><li>Teissi&#233;, J., Ramos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+electric+field+pulse+induced+long-lived+permeabilization+and+fusogenicity+in+cell+membranes.">Correlation between electric field pulse induced long-lived permeabilization and fusogenicity in cell membranes.</a> Biophys. J. 74, 1889-1898 (1998).</li></ol>

利害の衝突は宣言されません。

非特異的に、例えば、外部電界によっては、融合する細胞膜の能力は、生物学、バイオテクノロジー、医学研究のために重要です。このような非特異的な融合は、例えばモノクローナル抗体のような非常に貴重なハイブリッド細胞とその製品の生産を可能にし、[2]核融合の基本的メカニズムに関する情報を提供します。それが適切に異なる種類の細胞に調整できるので、電気融合は、潜在的に非常に効果的な方法です。密接な物理的接触で細胞を高電圧の電気パルスによって彼らの融合状態（核融合を起こしやすい）に持って来られるときに電気融合が達成されます。電気融合の効率は、電気融合過程の二つの部分に影響を与えるさまざまなパラメータに依存します。電気融合過程の最初の部分は、細胞間の緊密な物理的な接触の成果であり、これは別の方法で得られる[3-8]。密着法（コンフルエンスまで増殖する細胞）が原因で細胞間の大きなゾーンで自発的に確立されたセルの連絡先に効率的に使用できますが、それは多くの原子核で非常に大規模な融合細胞を生成します。我々は生き残るために可能性が高いと増殖する小さな細胞が（2〜5核をもつ）、、得られる改変遵守方法を、（図1）を使用している。細胞間の接触は、また実験[9]で使用される浸透圧処理により、細胞の浸透圧膨張の恩恵を受ける。電気融合プロセスの後半では、細胞膜の融合状態を達成することである。融合状態は、膜のelectropermeabilized状態（細胞が非特異的に、通常はそのままに膜を通過できない分子に透過処理したもの）とよく相関し、電気パルス（振幅、長さ、数と周波数）の同じパラメータによって支配される[10] 。最適なエレクトロポレーションに必要な電気的パラメータの値が[1]と電気融合が異なるセル間で異なると細胞の大きさとその生物学的特性に依存する。電気的パラメータは、このように融合を得るために、融合パートナーとして使用されるさまざまな細胞株に最適化する必要があります。

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		<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>CMRA</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>C34551</td>
<td>Cytosolic fluorescent dye</td>
</tr>
<tr>
<td>CMFDA</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>C7025</td>
<td>Cytosolic fluorescent dye</td>
</tr>
<tr>
<td>DMSO</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>D2650</td>
<td>&nbsp;</td>
<tr>
<td>DMEM </td>
<td>Reagent</td>
<td>Sigma-Aldrich </td>
<td>D5671</td>
<td>Dulbecco's modified Eagle's medium</td>
</tr>
<tr>
<td>Fetal calf serum</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F4135</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>G7513</td>
<td>&nbsp;</td>
<tr>
<td>crystacillin</td>
<td>Reagent</td>
<td>Pliva</td>
<td>625110</td>
<td>antibiotic</td>
</tr>
<tr>
<td>gentamicin</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>G1397</td>
<td>antibiotic</td>
</tr>
<tr>
<td>Hepes</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>H0887</td>
<td>&nbsp;</td>
<tr>
<td>KH2PO4</td>
<td>Reagent</td>
<td>Merck </td>
<td>A124873 927</td>
<td>&nbsp;</td>
<tr>
<td>KH2PO4</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>4248</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>M-8266</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>Reagent</td>
<td>Fluka</td>
<td>71382</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>Reagent</td>
<td>Merck</td>
<td>A154336 908</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>M2643</td>
<td>&nbsp;</td>
<tr>
<td>D-glucose</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>G8270</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>C4901</td>
<td>&nbsp;</td>
<tr>
<td>sucrose</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>16104</td>
<td>&nbsp;</td>
<tr>
<td>Electric pulse generator</td>
<td>Tool</td>
<td>Igea</td>
<td>&nbsp;</td>
<td>Cliniporator VITAE</td>
</tr>
<tr>
<td>Multiwell plate</td>
<td>Tool</td>
<td>TPP</td>
<td>92424</td>
<td>&nbsp;</td>
<tr>
<td>50 ml centrifuge tube</td>
<td>Tool</td>
<td>TPP</td>
<td>91050</td>
<td>&nbsp;</td>
<tr>
<td>15 ml centrifuge tube</td>
<td>Tool</td>
<td>TPP</td>
<td>91015</td>
<td>&nbsp;</td>
<tr>
<td>25 cm2 culture flask</td>
<td>Tool</td>
<td>TPP</td>
<td>90026</td>
<td>&nbsp;</td>
<tr>
<td>Electrodes</td>
<td>Tool</td>
<td>Custom made</td>
<td>&nbsp;</td>
<td>Pt/Ir</td>
</tr>		</tbody>
		</table>
		</div>

cell electrofusion visualized with fluorescence microscopy

細胞の電気融合は、ヒト​​の治療のためのハイブリッド細胞を調製するために使用することができる安全な、非ウイルス性および非化学的な方法です。電気融合は密着している細胞に短い高電圧の電気パルスのアプリケーションが含まれます。短期、高電圧の電気パルスのアプリケーションは、細胞の形質膜の不安定化を引き起こす。不安定化膜は、異なる分子のため、より透過性とは、近接する不安定化膜との融合にもなりやすいです。電気融合は、このように非常に異なる細胞の非特異的な融合を実現する便利な方法です。<em&gt; in vitroで</em&gt;。電場による不安定核、細胞膜を、得るために、それらの脂質二重層とその結果としてその細胞質のマージできるように密着している必要があります。このビデオでは、我々は細胞の効率的な電気融合を示しています<em&gt; in vitroで</em&gt;修正された接着法による。この方法では、細胞がウェルの表面にわずかに接続を許可されているので、その媒体を交換できるようになり、細胞はまだ彼らの球状の形状を保持。融合可視化は、異なる蛍光細胞トラッカーの色素を持つ細胞の細胞質のプレラベリングによって評価され、細胞の半分がオレンジ色CMRAと緑CMFDAで残りの半分で標識されています。核融合の利回りは、2つを掛けたすべてのセルの数で割った二重に蛍光細胞の数として決定されます。

Watch this Scientific Journal Video about Cell Electrofusion Visualized with Fluorescence Microscopy at JoVE.com

Cell Electrofusion Visualized with Fluorescence Microscopy

このビデオでは、細胞の効率的な電気融合を示しています<em&gt; in vitroで</em&gt;エレクトロポレーションおよび蛍光顕微鏡による融合細胞の可視化のその後の検出を使用して変更密着法による。

細胞の電気融合を蛍光顕微鏡で可視化

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

cell-electrofusion-visualized-with-fluorescence-microscopy

Research

JoVE Journal

Biology

14.0K ビュー.  University of Ljubljana. このビデオでは、細胞の効率的な電気融合を示しています in vitroでエレクトロポレーションおよび蛍光顕微鏡による融合細胞の可視化のその後の検出を使用して変更密着法による。

ビデオ: Cell Electrofusion Visualized with Fluorescence Microscopy

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

蛍光顕微鏡による可視化単分子のDNAの複製

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

生物学

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

Hui Zhang and Niko G. Gubernator contributed equally to this work.

A new means to measure neurotransmission optically using fluorescent dopamine analogs.

個々のシナプス前終末でのドーパミン放出はFFNsと可視化

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNAトランスフェクション後の有糸分裂のタイムラプスイメージング

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

生細胞におけるストア作動性カルシウムエントリの蛍光ベースの測定：培養癌細胞から骨格筋線維に

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

分裂酵母の定量的なライブセル蛍光-顕微鏡解析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

タバコプロトプラストと葉で二分子蛍光相補性によって可視化し、タンパク質間相互作用

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

単一のビリオン原子間力顕微鏡と超解像蛍光イメージングのサンプル調製

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

蛍光顕微鏡でストロミュール頻度を可視化

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

微生物細胞の成長に不可欠なプロセスを遵守する細胞蛍光顕微鏡をライブします。

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...