私たちは、ケージフルオレセインを作る際に助けのためにダンカーリンに感謝します。さらに、我々は次の資金源に感謝したい：JACに発生生物学の訓練助成金のためのヴァンダービルト大学医学部のプログラムを、とNIH HD054534to JTG。

1。リテーナフルオレセインデキストランの合成<ol><li>うち3.5測定 - アミノデキストランの4 mgをしCMNB -ケージフルオレセインSEの1mgを含有するInvitrogen社が提供する着色チューブに加える。私たちの手でこの比率は、デキストランあたり〜2.5色素分子の平均的な負荷を与えます。 </li><li>チューブに0.1 MのNa 2 B 4 O 7（ホウ酸ナトリウム）バッファーを500μLを加える。 </li><li>デキストランとケージフルオレセインを溶解するために30秒間のキャップと渦。 </li><li>ボルテックスミキサーで一晩反応してみましょう。 </li><li> Zebaスピンカラムの底部の閉鎖をねじり切るとキャップを緩めます。フェルトペンでカラム上のドットをマーク。 15 mLコニカルチューブの場所の列。 </li><li>ローターの中心が直面している点で2分間1,000 × gで遠心する。圧縮後すぐにカラムを使用してください。 </li><li>新しい15 mLコニカルチューブに圧縮列を置きます。 </li><li>プールの反応混合物と圧縮カラムの樹脂床の中心に移転。キャップを交換してください。 </li><li>ローターの中心が直面している点で2分間1,000 × gで遠心する。スピンした後、溶離液とカラムの上部には、色の黄色のほぼ同じ色合いになります。 </li><li> 1.5 mLのマイクロ遠心チューブに黄色の溶離液（〜350μL）を転送する。 </li><li> speedvacに乾燥するまで凍結乾燥。箔とspeedvacをカバーしています。 </li><li>その結果、再サスペンド〜水中の1％の最終濃度w / vのに淡黄色泡状の2-3ミリグラム</li><li>箔で覆われたチューブ内で-20℃でこのソリューションを保管してください。それはアリコートに、繰り返し凍結融解を防ぐためにデキストラン溶液は良い考えです。 </li></ol> 2。ゼブラフィッシュ胚に入りフルオレセインデキストランの注入<ol><li> 0.2MのKClでケージフルオレセインデキストラン1：05または1:10の1％（w / v）の株式を希釈する。 </li><li> 0.5注入 - ケージフルオレセイン溶液の1.0nLを圧力注入器を使用して1細胞期胚の卵黄に。一度注入、胚のクラッチは、° Cの非特異的アンケージングを減らすために28.5で暗所に保管する必要があります。 </li><li>手動で胚からchorionsを削除します。 </li><li> 4パーセントtricaineの胚を麻酔。 </li><li>胚はその後、卵の水で覆われ、35mmx10mmのペトリ皿に0.8％アガロースでマウントされます。 </li><li>少なくとも15分間50℃のヒートブロックにアガロースを融解平衡化させます。 </li><li>ガラスピペットを用いて、水の最小量の内の1つの胚を策定し、アガロースノートにそれを解放します。それは前に避けるために、チューブ内に胚を置くことに約30秒間手の中でアガロースのチューブを冷却することが重要です。胚を殺す。 </li><li>約50μLアガロースのと一緒に、チューブから胚をピペットとペトリ皿の中央に内容を取り出します。 </li><li>すぐに面が上になるように向きuncagedする細胞とプラスチックのスレッドを使用して、胚注：クールとアガロースは、速やかに胚を配向させると性急である。アガロースがリジッドになった後に操作した場合の胚が損傷することができます。 </li><li>アガロースが完全に固化（これは数分かかります）、4％tricaineと0.003％のPTUを含む卵を水で完全に皿3分の2を埋めることができます。 </li></ol> 3。フルオレセインデキストランのレーザーアンケージング<ol><li>アンケージングのために使用される顕微鏡は40倍の水浸対物レンズを持っている必要があります。 </li><li>我々は365nmの色素細胞と70％/ 30％のスプリットダイクロイックミラーで光計測器レーザーを使用してください。 </li><li>アンケージングする前に、レーザーは、客観的かつ適切な電力の減衰と同焦点にしなければならない。 </li><li>レーザーを当てるために、客観的で、ミラー化されたスライドガラスを配置し、スライドの傷が見えるようになるまで目標を置いています。 </li><li>レーザーは単一パルスとミラーではっきりと目に見える傷を作り出すことができるまで、レーザー減衰器を調整します。 </li><li>客観的なフォーカスを調整し、ダウン。目標の焦点が合っていないときにレーザーの傷を作ることができれば、レーザーの焦点を調整¼上下に回すか。目的がフォーカスされているときのみレーザーがミラーをスクラッチできるようになるまで繰り返します。 </li><li>場所はuncagedする領域上で客観的かつ焦点の下胚をマウント。 </li><li>鳥かごから出すために、目的の細胞に焦点を当て、2〜3 Hzでそれを10〜20倍のパルスを与える。 </li><li>鉗子で離れてアガロースを剥離し、0.003％のPTUを含む卵の水でそれらを置くことによって、胚アンケージング後、フリー。それらが修復されるまで、胚は遮光ボックスで開発しましょう​​。 </li></ol> 4。 Uncagedフルオレセインデキストランの抗体ラベリングと検出<ol><li>抗体固定液に胚を修正4℃、室温（RT）または一晩（O / N）で2〜4時間℃で（4％パラホルムアルデヒドを、0.3mmのCaCl 2で 、8％のショ糖を、1Xのリン酸緩衝生理食塩水、pHが7.3バッファ）光タイトボックスまたはすべてのラベリングのステップのためのアルミホイルに包まれたチューブに胚を保持。 </li><li>固定液を除去し、100％MeOHで置き換え、室温で10分間放置し、MeOHとreplumの複数形を削除する新鮮な100％のMeOHでCE。 </li><li>少なくとも30分間、-20℃でメタノールのストア注：。エピトープが低下し始める前に、最大2週間までMeOHに格納することができます。 </li><li> 75パーセントMeOH/1Xリン酸塩の室温で5分間洗浄した胚を再水和するが0.1％のTween 20（1X PBSTw）と緩衝生理食塩水、次いで50％MeOH/50％の1XPBTw、、25％MeOH/75％1X PBSTw。 </li><li> 1XPBSTwで5分間3回洗浄する。 </li><li>胚は、&gt; 24時間経過した場合、10μgの/ 1XPBSTw mLのプロテイナーゼKで胚をpermiabilize。 Permiabilization時間は、ステージに依存しています。胚は、24および48時間後に受精（HPF）の間にある場合プロテイナーゼKで5分で十分です。より多くの時間は、古い幼虫の場合必要になることがあります。室温で20分間抗体固定液で再修正</li><li>室温で1XPBSTwで5分間5回洗浄する。 </li><li>室温で1-2時間（1XPBS、0.1％トリトンX100、10％ヒツジ血清、10％ウシ血清アルブミン、1％ジメチルスルホキシド）ブロックの溶液中でインキュベートする。 </li><li>ブロッキング溶液に一次抗体（抗フルオレセインを加えた細胞型特異的マーカーに対する抗体）を希釈する。 </li><li> 4℃でインキュベート胚O / N °一次抗体を含む溶液中でC。 </li><li> 1XPhosphateで20分間4回洗浄すると、室温で0.1％トリトンX100（PBSTx）と緩衝生理食塩水。 </li><li>ブロッキング溶液で蛍光標識二次抗体を（通常は1:300）に希釈する。 </li><li> 4℃でインキュベートするO / N °二次抗体を含む溶液中でC。 </li><li> RTで1XPBSTxで20分間4回洗浄。 </li><li>室温で少なくとも2時間は50％glycerol/50％の1XPBSの明確な胚は、その後、グリセロール/ PBSの混合物を削除し、100％のグリセロールを追加し、4℃でO / Nを放置℃を</li></ol> 5。代表的な結果： 免疫標識した後、（Figure.1）uncagedされた細胞で染色の豊かな領域があるはずです。それはフルオレセインの自発アンケージングによる、&gt; 48時間古いゼブラフィッシュで雑音比に比較的高い信号を見ることも珍しくありません。 <img src="/files/ftp_upload/2672/2672fig1.jpg" alt="図1" /> ゼブラフィッシュ松果体複合体の図1。リネージュラベリング。 24でHPF、foxd3で示される前松果体原基の一部、：。GFPの発現は、紫外レーザーパルスを用いてuncagedした）48hpfすることにより、セルの左サイドクラスタがparapinealと呼ばれる（赤い丸）uncagedから浮上しているドメイン（白丸）。B）Uncagedフルオレセインの信号がparapineal（赤い円に富んでいる）と松果体（白丸）。

<ol>
	<li>Hatada, Y., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fate+map+of+the+epiblast+of+the+early+chick+embryo.">A fate map of the epiblast of the early chick embryo.</a> Development. 120, 2879-2889 (1994).</li><li>Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+of+the+blastomeres+of+the+32-cell-stage+Xenopus+embryo.">Fates of the blastomeres of the 32-cell-stage Xenopus embryo.</a> Dev Biol. 122, 300-319 (1987).</li><li>Wetts, R., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+Precursors+Can+Give+Rise+to+All+Major+Cell-Types+of+the+Frog+Retina.">Multipotent Precursors Can Give Rise to All Major Cell-Types of the Frog Retina.</a> Science. 239, 1142-1145 (1988).</li><li>Wetts, R., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+fluorescent+tracers+to+study+neural+cell+lineages.">Microinjection of fluorescent tracers to study neural cell lineages.</a> Development. , 1-8 (1991).</li><li>Woo, K., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Order+and+coherence+in+the+fate+map+of+the+zebrafish+nervous+system.">Order and coherence in the fate map of the zebrafish nervous system.</a> Development. 121, 2595-2609 (1995).</li><li>Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc Natl Acad Sci U S A. 99, 12651-12656 (2002).</li><li>Sato, T., Takahoko, M., Okamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HuC:Kaede,+a+useful+tool+to+label+neural+morphologies+in+networks+in+vivo.">HuC:Kaede, a useful tool to label neural morphologies in networks in vivo.</a> Genesis. 44, 136-142 (2006).</li><li>Wiedenmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EosFP,+a+fluorescent+marker+protein+with+UV-inducible+green-to-red+fluorescence+conversion.">EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.</a> Proc Natl Acad Sci U S A. 101, 15905-15910 (2004).</li><li>Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Fluorescent Probes and Research Chemicals. , 707-711 (2002).</li><li>Kozlowski, D. J., Murakami, T., Ho, R. K., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+cell+movement+and+tissue+patterning+in+the+zebrafish+embryo+revealed+by+fate+mapping+with+caged+fluorescein.">Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein.</a> Biochem Cell Biol. 75, 551-562 (1997).</li><li>Kozlowski, D. J., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivatable+(caged)+fluorescein+as+a+cell+tracer+for+fate+mapping+in+the+zebrafish+embryo.">Photoactivatable (caged) fluorescein as a cell tracer for fate mapping in the zebrafish embryo.</a> Methods Mol Biol. 135, 349-355 (2000).</li><li>Russek-Blum, N., Nabel-Rosen, H., Levkowitz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+fate+map+of+the+zebrafish+diencephalon.">High resolution fate map of the zebrafish diencephalon.</a> Dev Dyn. 238, 1827-1835 (2009).</li><li>Summers, R. G., Piston, D. W., Harris, K. M., Morrill, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+orientation+of+first+cleavage+in+the+sea+urchin+embryo,+Lytechinus+variegatus,+does+not+specify+the+axes+of+bilateral+symmetry.">The orientation of first cleavage in the sea urchin embryo, Lytechinus variegatus, does not specify the axes of bilateral symmetry.</a> Dev Biol. 175, 177-183 (1996).</li></ol>

利害の衝突は宣言されません。

説明したように、このプロトコルは、マイクロインジェクション、顕微鏡、および免疫蛍光の一般的に使用される技術に基づいて構築されているゼブラフィッシュでは比較的速い系統の標識法を提供する。我々は、レーザーは、ローカライズされた形でフルオレセインを刑務所から出すために最も効率的かつコスト効果の高い方法であることがアンケージングことを発見した。このメソッド?...

A correction was made to <a href="http://www.jove.com/details.php?id=2672">Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran</a>. There was an error in the authors, Ilya A. Shestopalov and James K. Chen, names. The author's names have been corrected to: Ilya A. Shestopalov and James K. Chen instead of: Ilya Shestopalov and James Chen

A correction to the article Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran was made.

Erratum: Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

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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>CMNB-caged fluorescein SE</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C20050</td>
<td>&nbsp;</td>
<tr>
<td>Aminodextran, 10kDa</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D1860</td>
<td>&nbsp;</td>
<tr>
<td>Zeba desalt spin columns</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>89889</td>
<td>&nbsp;</td>
<tr>
<td>goat anti-fluorescein antibody</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A11095</td>
<td>&nbsp;</td>
<tr>
<td>Alexa Fluor 633 Donkey anti-goat antibody</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A21082</td>
<td>&nbsp;</td>
<tr>
<td>35mmx10mm petri dishes</td>
<td>&nbsp;</td>
<td>Becton-Dickinson</td>
<td>351008</td>
<td>&nbsp;</td>
<tr>
<td>Upright compound microscope with 40x water immersion objective</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>DM6000B</td>
<td>&nbsp;</td>
<tr>
<td>MicroPoint Manual Laser Illumination System</td>
<td>&nbsp;</td>
<td>Photonic Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phenylthiourea (PTU)</td>
<td>&nbsp;</td>
<td>Alfa Aesar</td>
<td>L06690</td>
<td>&nbsp;</td>
<tr>
<td>Tricaine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E10521</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>03115836991</td>
<td>&nbsp;</td>
<tr>
<td>Plastic thread</td>
<td>&nbsp;</td>
<td>Any sewing supply store</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Agarose</td>
<td>&nbsp;</td>
<td>Research Products International</td>
<td>A20090</td>
<td>&nbsp;</td>
<tr>
<td>SpeedVac</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>Savant SPD131DDA</td>
<td>&nbsp;</td>
<tr>
<td>Vortexing mixer</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>Vortex Genie 2</td>
<td>&nbsp;</td>
<tr>
<td>Pressure injector</td>
<td>&nbsp;</td>
<td>Applied Scientific Instrumentation</td>
<td>MPPI-3</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

lineage labeling of zebrafish cells with laser uncagable fluorescein dextran

発生生物学の中心問題は、未分化前駆体から生じるとして、脊椎動物に存在する無数の細胞型の起源を推定することです。研究者は、DIIラベル1とモデルシステムの開発の後期段階で把握細胞の運命にトレーサブルな酵素の圧力注入2のような系統の標識の様々な方法を採用している。ゼブラフィッシュ（ ゼブラフィッシュ ）の最初の運命のマップは、胚の離散的な領域で単一の細胞に、そのようなローダミンデキストランとして、蛍光色素のイオン導入注入によって組み立てられ、時間3-5を介して標識細胞の運命をトレースしていた。効果的な一方で、これらの方法は技術的に要求しており、また一般的にゼブラフィッシュラボでは見られない特殊な装置を必要としている。最近、紫外線にさらされると不可逆的に緑色から赤色蛍光への切り替えなどイーオスや楓などのphotoconvertable蛍光タンパク質は、、、ゼブラフィッシュ6-8の使用の増加を見ている。ゼブラフィッシュ胚や遺伝子導入の相対的な容易さの光学的透明度は、系列ラベリングのためのこれらの特に魅力的なツールを作られているし、in vivoで7に細胞の移動を観察する。それらのユーティリティにもかかわらず、これらのタンパク質は、色素を介した系統の標識方法と比べて不利な点がいくつかある。最も重要な我々はこれらのタンパク質の光変換の間に高い3次元分解能を得るに見ている難しさです。この光の中で、おそらくゼブラフィッシュの系統の標識のための解像度と使いやすさの最適な組み合わせは、ケージフルオレセインデキストランの使用、消光グループにバインドされている蛍光色素になりますそのマスクの蛍光9。染料は、その蛍光または免疫検出の可視化を可能にする、レーザーや水銀ランプからの紫外光を使用して特定のセル内（消光グループから解放さ）&quot;uncaged&quot;することができます。イオン導入の方法とは異なり、ケージフルオレセインは、標準的な注入装置を挿 ​​入することができ、ピンホール10を装備した蛍光顕微鏡でuncaged。さらに、フルオレセインに対する抗体だけuncagedフォームを検出し、エピトープは、固定も11生き残っています。最後に、ケージフルオレセインは、2光子顕微鏡は、12,13を採用している場合は特に、非常に高い3次元分解能をアクティブにすることができます。このプロトコルは、ケージフルオレセインとレーザーのアンケージングによって系統のラベリングの方法を説明します。 Subsequenctly、uncagedフルオレセイン抗体で標識することによりGFPなどの他のエピトープと同時に検出される。

Watch this Scientific Journal Video about Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran at JoVE.com

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

このプロトコルは、uncagedフルオレセインからの信号を増幅するために免疫標識全体のマウントが続くケージフルオレセインのUV -アンケージングを、使用してセルゼブラフィッシュ胚の小グループの運命にラベルを付けたり、トレースする方法を区別します。

レーザーUncagableフルオレセインデキストランでゼブラフィッシュ細胞の系統ラベリング

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

lineage-labeling-zebrafish-cells-with-laser-uncagable-fluorescein

Research

JoVE Journal

Biology

13.8K ビュー.  Vanderbilt University. このプロトコルは、uncagedフルオレセインからの信号を増幅するために免疫標識全体のマウントが続くケージフルオレセインのUV -アンケージングを、使用してセルゼブラフィッシュ胚の小グループの運命にラベルを付けたり、トレースする方法を区別します。

ビデオ: Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

光イメージングによる非侵襲的検出のための蛍光色素で標識幹細胞

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

生物学

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

代謝標識とBioorthogonalクリックケミストリーによるゼブラフィッシュ胚でのイメージンググリカン

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

腎上皮再生を研究するためにゼブラフィッシュの前腎のレーザーアブレーション

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking1,2 but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases3.
Yet their existence is still a matter of controversy4,5. Indeed, lipid raft size has been estimated to be around 20 nm6, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized7,8. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells.
Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts9,10. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models11.
FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently12. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change12.

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS

A technique to probe the lipid raft partitioning of fluorescent proteins at the plasma membrane of living cells is described. It takes advantage of the disparity in diffusion times of proteins located inside or outside of lipid rafts. Acquisition can be performed dynamically in control conditions or after drug addition.

脂質ラフトの決定は、蛍光相関分光法（FCS）による生細胞の蛍光タグ付きプローブの分割

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

マウス造血幹細胞およびリネージュコミット前駆細胞の表現型の解析と分離

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

ゼブラフィッシュ胚骨格筋のレーザー招いた傷害

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

ゼブラフィッシュにおける腎毒素マイクロインジェクションは急性腎障害をモデルに

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Continuous-wave Thulium Laser for Heating Cultured Cells to Investigate Cellular Thermal Effects

An original method to heat cultured cells using a 1.94 &#181;m continuous-wave thulium laser for biological assessment is introduced here. Thulium laser radiation is strongly absorbed by water, and the cells at the bottom of the culture dish are heated through thermal diffusion. A laser fiber with a diameter of 365 &#181;m is set about 12 cm above the culture dish, without any optics, such that the laser beam diameter is almost equivalent to the inner diameter of the culture dish (30 mm). By keeping a consistent amount of culture medium in each experiment, it is possible to irradiate the cells with a highly reproducible temperature increase.
To calibrate the temperature increase and its distribution in one cell culture dish for each power setting, the temperature was measured during 10 s of irradiation at different positions and at the cellular level. The temperature distribution was represented using a mathematical graphics software program, and its pattern across the culture dish was in Gaussian form. After laser irradiation, different biological experiments could be performed to assess temperature-dependent cell responses. In this manuscript, viability staining (i.e., distinguishing live, apoptotic, and dead cells) is introduced to help determine the threshold temperatures for cell apoptosis and death after different points in time.
The advantages of this method are the preciseness of the temperature and the time of heating, as well as its high efficiency in heating cells in a whole cell culture dish. Furthermore, it allows for study with a wide variety of temperatures and time durations, which can be well-controlled by a computerized operating system.

An original experimental setup for heating cells in a culture dish using 1.94 &#181;m continuous-wave laser radiation is introduced here. Using this method, the biological responses of retinal pigment epithelial (RPE) cells after different thermal exposures can be investigated.

培養細胞を加熱して細胞の熱作用を調べるための連続波型ツリウムレーザー

An original method to heat cultured cells using a 1.94 &#181;m continuous-wave thulium laser for biological assessment is introduced here. Thulium laser ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

可視化と系統特異的マーカーと間葉系細胞脂肪細胞の微分の潜在性の定量化

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

げっ歯類の頭部に定義された加速度を生成するための受動的頭部運動の適用

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...