と医学のアルバートアインシュタイン大学からスタートアップ資金、この作品は、部分的に国立衛生研究所（RDSへ3U54AI057158 - 06S1 PWにGM093282）によってサポートされていました。

1。卵の回収とDechorionation <ol><li>収集と35mmのペトリ皿にゼブラフィッシュ卵を転送、できるだけ多くの水のように削除してから、E3胚の培地で1 mg / mlのProneaseのE（5 mMのNaCl、0.17 mMの塩化カリウム、0.33 mMの塩化カルシウム2 · 2H 2 O、0.33 mMのマグネシウムを追加する絨毛膜を消化するために4、pH = 7.4）で。 </li><li> 3-5分後、魚の水（1リットル当たり60mgの&quot;インスタントオーシャン&quot;蒸留H 2 O）で満たされたビーカーに皿をマージし、穏やかにビーカーに卵を移すと、卵が水の中に&quot;落ちる&quot;ことができます。 </li><li>魚の水と卵を3回すすいでください。ほとんどの卵は、それらの絨毛膜から解放されます。 </li><li>ファイアーポリッシュガラスパスツールピペットを用いて、E3胚の媒体が充填さアガロースコートしたペトリ皿にdechorionated卵を移す。 </li></ol> 2。 GDP - FucAlとマイクロインジェクション<ol><li>報告されたプロトコル7次の注入の料理を準備します。 </li><li> E3胚の媒体が充填注入皿にdechorionated卵を移す。 </li><li>針を準備し、2μL注入液で読み込みます。このソリューションでは、トレーサーまたは0.2 M KClの中のフェノールレッドローディング色素（0.1％w / v）のようchemoenzymatically 8とのAlexa Fluor ® 594 -デキストラン（5％w / v）のいずれかを合成したGDP - FucAl 20mMのが含まれています。ネガティブコントロールとして、注射液のGDP -フコースとGDP - FucAlを交換してください。 </li><li>針を破ると1 NL降下9を得るために射出圧力および持続時間を調整する。 </li><li>どちらのソリューションの1 NLと卵を注入する。 </li><li> E3胚の媒体が充填さアガロースコートしたペトリ皿に卵を移す。 </li><li> 28 ° Cで卵をインキュベートし、受精後3〜4時間以内に未受精卵を削除します。 </li></ol> 3。 BTTES - Cu系（I）触媒をクリックして化学反応<ol><li>胚は発達段階（例えば後期原腸胚、組織のセグメンテーションと早期の幼虫）、コート、アガロースと96ウェルプレートの基盤を求め達した時点で。 </li><li> 4μLのAlexa Fluor ® 488アジド（H 2 O、2.5 mMストックから）、2μLBTTES - 4 · 6時01分、複雑な、そしてミックスに軽く振る加え、次いで、各ウェルに92μLE3胚の培地を追加。 </li><li>よくファイアーポリッシュガラスパスツールピペットを用いてクリックケミストリー試薬を含むに胚を移す。各ウェルは、5つの胚よりも少ないが含まれている必要があります。 </li><li>クリック反応3を開始するには2.5μL、新たに調製したアスコルビン酸ナトリウムを（H 2 O中の100mMのストックから）を追加します。各試薬の終濃度：のAlexa Fluor - 488アジ：100μM;のCuSO 4：50μM; BTTES：300μM、アスコルビン酸ナトリウム：2.5 mMの。 </li><li> 3分後、100μLE3の胚の培地で直ちに希釈して反応をクエンチするために、2μLバソクプロインスルホン酸（H 2 Oに50 mMストック）、生体適合性の銅キレート剤を加える。 </li><li>ガラスシャーレに胚を移し、15 mLのE3胚の培地で処理胚を2回洗浄する。 </li></ol> 4。イメージング<ol><li>マテックのガラス底マイクロウェル皿に超低融点アガロース（E3胚の培地では1.2％（w / v）の）の低下を置き、アガロースドロップに胚を置く。 </li><li>背側または横方向に胚を置き、アガロースドロップを固化するために氷上で5分間皿を置きます。それはアガロースドロップをカバーするまで、皿にそっとE3胚の培地を追加。 </li><li>蛍光と明視野の画像を順次共焦点顕微鏡を用いて取得されます。すべての胚の画像は、5μmのステップ間隔を使用して取得されます。複合数値は、ImageJのソフトウェアを用いて調製されています。 </li></ol> 5。代表的な結果図1は、私たちの二段階ラベリング戦略のワークフローを示しています。図2は、9.5時間後に受精（HPF）でBTTES介したCuAAC経由のゼブラフィッシュ胚のラベルを示しています。 BTTESは、トリス（トリアゾリルメチル）アミン系配位子です。それはその場生成される銅（I）との調整をするときに劇的にCuAAC加速、と明らかに毒性のな​​い生活システムで急速に付加環化反応を促進する。すぐに3分のクリック反応を以下、我々は、GDP - FucAl治療胚（図2、左のパネル）の堅牢なラベルを検出することができます。唯一のバックグラウンド蛍光は、GDP -フコース（図2、右側のパネル）にマイクロインジェクション制御の胚のために検出されます。 <img src="/files/ftp_upload/2686/2686fig1.jpg" alt="図1" /> 図1ゼブラフィッシュ胚の包絡層にフコシル化糖鎖を標識する戦略。 <img src="/files/ftp_upload/2686/2686fig2.jpg" alt="図2" /> 図2。BTTES - Cu系（I）触媒クリックケミストリーを介してゼブラフィッシュ胚発生におけるフコシル化糖鎖のin vivoイメージング 。 1細胞期のゼブラフィッシュの胚は、GDP - FucAlの単回投与でマイクロインジェクションし、開発を許可されます9.5 HPFのop。胚はその後BTTES - Cu系（I）でのAlexa Fluor ® 488アジドの触媒と反応させる。反応胚は、共焦点顕微鏡を用いて画像化している。のAlexa Fluor ®蛍光488（上部パネル）の最大強度のz -投影画像、ブライトフィールド（下のパネル）。スケールバー：100μmである。 トラブルシューティング<table border="1"><tbody><tr><td>問題が</td><td>原因と</td><td>救済策</td></tr><tr><td>胚は不健康に見える</td><td>マイクロインジェクションのソリューションは、汚染物質が含まれています</td><td>ヌクレオチド糖の純度は85％以上でなければなりません。あなたのソースを確認してください。 </td></tr><tr><td>反応の後に表示は特にない</td><td>銅濃度は、以下の30μmである</td><td>銅の濃度が30μMのときに反応速度が著しく低下するとして、胚を追加する際に過剰E3胚の培地を加えることにより反応液を希釈しないように注意してください。 </td></tr><tr><td>胚は、反応後に死ぬ</td><td>胚の不適切な取り扱い</td><td>ピペットは、ファイアーポリッシュであり、反応容器を0.5％アガロースの非常に薄い層でコーティングされていることを確認します。 </td></tr><tr><td>画像がむらに見える</td><td>試薬は適切に胚を追加する前に混合されていません</td><td>試薬のほかの推奨順序に従ってください、とクリックケミストリー試薬が適切に胚を追加する前に混合されていることを確認してください。 </td></tr><tr><td>画像は、胚が損傷を受けている表示</td><td>反応中に激しく振盪損害胚</td><td>胚が溶液では1回、ゆっくりと10秒未満のために振る。 </td></tr><tr><td></td><td></td><td></td></tr></tbody></table>

<ol>
	<li>Laughlin, S. T., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+glycome.">Imaging the glycome.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 12-17 (2009).</li><li>Baskin, J. M., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioorthogonal+click+chemistry:+Covalent+labeling+in+living+systems.">Bioorthogonal click chemistry: Covalent labeling in living systems.</a> Qsar Comb. Sci. 26, 1211-1219 (2007).</li><li>Soriano del Amo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatible+copper(I)+catalysts+for+in+vivo+imaging+of+glycans.">Biocompatible copper(I) catalysts for in vivo imaging of glycans.</a> J. Am. Chem. Soc. 132, 16893-16899 (2010).</li><li>Chang, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+labeling+of+sialic+acids+in+living+animals+with+alkynyl+sugars.">Metabolic labeling of sialic acids in living animals with alkynyl sugars.</a> Angew. Chem. Int. Ed. 48, 4030-4033 (2009).</li><li>Laughlin, S. T., Baskin, J. M., Amacher, S. L., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+membrane-associated+glycans+in+developing+zebrafish.">In vivo imaging of membrane-associated glycans in developing zebrafish.</a> Science. 320, 664-667 (2008).</li><li>Baskin, J. M., Dehnert, K. W., Laughlin, S. T., Amacher, S. L., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+enveloping+layer+glycans+during+zebrafish+early+embryogenesis.">Visualizing enveloping layer glycans during zebrafish early embryogenesis.</a> Proc. Natl. Acad. Sci. U. S. A. 107, 10360-10365 (2010).</li><li>Kemp, H. A., Carmany-Rampey, A., Moens, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+chimeric+zebrafish+embryos+by+transplantation.">Generating chimeric zebrafish embryos by transplantation.</a> J. Vis. Exp. , (2009).</li><li>Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoenzymatic+synthesis+of+GDP-L-fucose+and+the+Lewis+X+glycan+derivatives.">Chemoenzymatic synthesis of GDP-L-fucose and the Lewis X glycan derivatives.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 16096-16101 (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <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+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J. Vis. Exp. , (2009).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> THE ZEBRAFISH BOOK, A guide for the laboratory use of zebrafish (Danio rerio). , (2007).</li><li>Kimmel, C. B., Warga, R. M., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+organization+of+the+zebrafish+fate+map.">Origin and organization of the zebrafish fate map.</a> Development. 108, 581-594 (1990).</li></ol>

利害の衝突は宣言されません。

<p class="jove_content"&gt;イメージング生体分子<em&gt; in vivoで</em&gt;母国の環境でそれらの生物活性の重要な洞察を提供する。このビデオでは、我々はゼブラフィッシュ胚の包絡層におけるフコシル化糖鎖の標識がGDP - FucAlとBTTESを介した生体適合性CuAACを介して第二段階の蛍光団の共役による1細胞期胚をマイクロインジェクションすることによって実現される方法を示しています<sup&gt; 3</sup&gt;。堅牢な標識は、2〜3?...

<table border="1"><tbody><tr><td>試薬の名前</td><td>会社</td><td>カタログ番号</td></tr><tr><td>硫酸銅（II）五水和物</td><td>シグマアルドリッチ</td><td> 203165 </td></tr><tr><td>のAlexa Fluor ® 488アジド</td><td>インビトロジェン</td><td> A10266 </td></tr><tr><td>デキストラン、Alexa Fluor 594の</td><td>インビトロジェン</td><td> D - 22913 </td></tr><tr><td> （+）- L -アスコルビン酸ナトリウム</td><td>シグマアルドリッチ</td><td> A7631 </td></tr><tr><td> Bathocuproinedisulfonic酸</td><td>アクロスオーガニック</td><td> 164060010 </td></tr><tr><td>ガラス底マイクロウェル皿</td><td>マテック</td><td> P35G - 1.5 - 14 - C </td></tr></tbody></table>

imaging glycans in zebrafish embryos by metabolic labeling and bioorthogonal click chemistry

in vivoでのイメージングの糖鎖は、最近、アジドまたはアルキンタグ付き単1、2細胞または生物を処理することにより、bioorthogonal化学レポーターの戦略を使用して有効になっている。糖鎖生合成機構によって処理される変更された単糖類は、細胞表面の複合糖質に組み込まれています。 bioorthogonalアジドまたはアルキンタグは、可視化用蛍光プローブと共有結合を許可する、または濃縮およびglycoproteomic分析のための親和性プローブ​​と。のエンベロープ層のフコシル化糖鎖を標識する）GDP - 5 - alkynylfucose（GDP - FucAl）、2と1細胞期胚の1）マイクロインジェクション：このプロトコルは、一般的に含むゼブラフィッシュ胚におけるフコシル化糖鎖の非侵襲的イメージングに使用する手順について説明します。共焦点顕微鏡3で生体適合性銅（I）触媒によるアジド-アルキン付加環（CuAAC）、及び3）イメージングを経由してアジド共役フルオロフォアとゼブラフィッシュ胚。方法は、ここで説明容易にゼブラフィッシュを開発する際に、他の生物において、糖鎖、シアル酸4とN -アセチルガラクトサミン5を含むなどの糖鎖、6の他のクラスを可視化するために拡張することができます。

Watch this Scientific Journal Video about Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry at JoVE.com

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

ゼブラフィッシュ胚におけるアルキンタグ付き糖鎖の迅速、非侵襲的、および堅牢なラベリングを可能にするクリックケミストリーベースの方法が記載されている。後期原腸形成の段階におけるゼブラフィッシュ胚の包絡層におけるフコシル化糖鎖は、本研究では画像化した。

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 ...

imaging-glycans-zebrafish-embryos-metabolic-labeling-bioorthogonal

Research

JoVE Journal

Biology

13.5K ビュー.  Albert Einstein College of Medicine, Yeshiva University. ゼブラフィッシュ胚におけるアルキンタグ付き糖鎖の迅速、非侵襲的、および堅牢なラベリングを可能にするクリックケミストリーベースの方法が記載されている。後期原腸形成の段階におけるゼブラフィッシュ胚の包絡層におけるフコシル化糖鎖は、本研究では画像化した。

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

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

ゼブラフィッシュ胚におけるmRNAおよびモルホリノアンチセンスオリゴヌクレオチドのマイクロインジェクション。

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

生物学

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

ライブゼブラフィッシュの胚における二光子軸索切断とタイムラプス共焦点イメージング

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

移植によるキメラゼブラフィッシュ胚の生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

キメラゼブラフィッシュ胚における網膜と脳の開発のライブイメージングのためのGFP発現割球の移植

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

リビングでのイメージング、細胞の形状の変更ショウジョウバエ胚

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

代謝標識とフローサイトメトリーで細胞周期の進行速度を測定

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

ゼブラフィッシュ胚の代謝プロファイル解析

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

活性化カスパーゼ3を検出するために、ホールマウント免疫蛍光法によりゼブラフィッシュ胚におけるアポトーシスの分析

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

ゼブラフィッシュ胚における酸化ストレスの分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

ゼブラフィッシュ胚における組織特異的蛍光の半自動イメージング

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...