GFP -β-アクチンプラスミドは、寛大に博士ウェインオアー、病理学のLSUHSC - S部門によって提供された、と博士はベッキーWorthylake、薬理学のLSUHSC - NO学科の研究室で増幅した。この作品は、国立衛生研究所（P20 RR - 018766）とアメリカ心臓協会（05835386N）からの補助金によって支えられている。

<ol>
	<li>Spindler, V., Schlegel, N., Waschke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+GTPases+in+control+of+microvascular+permeability.">Role of GTPases in control of microvascular permeability.</a> Cardiovasc. Res. 87, 243-253 (2010).</li><li>Wojciak-Stothard, B., Ridley, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+GTPases+and+the+regulation+of+endothelial+permeability.">Rho GTPases and the regulation of endothelial permeability.</a> Vascul. Pharmacol. 39, 187-199 (2002).</li><li>Yuan, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+pathways+in+enhanced+microvascular+permeability.">Signal transduction pathways in enhanced microvascular permeability.</a> Microcirculation. 7, 395-403 (2000).</li><li>Birukov, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+GTPases+in+mechanosensitive+regulation+of+endothelial+barrier.">Small GTPases in mechanosensitive regulation of endothelial barrier.</a> Microvasc. Res. 77, 46-52 (2009).</li><li>Duran, W. N., Sanchez, F. A., Breslin, J. W., Tuma, R. F., Duran, W. N., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcirculatory+Exchange+Function.">Microcirculatory Exchange Function.</a> Handbook of Physiology: Microcirculation. , 81-124 (2008).</li><li>Hu, Y. L., Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+motion+of+paxillin+on+actin+filaments+in+living+endothelial+cells.">Dynamic motion of paxillin on actin filaments in living endothelial cells.</a> Biochem. Biophys. Res. Commun. 357, 871-876 (2007).</li><li>Borm, B., Requardt, R. P., Herzog, V., Kirfel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+ruffles+in+cell+migration:+indicators+of+inefficient+lamellipodia+adhesion+and+compartments+of+actin+filament+reorganization.">Membrane ruffles in cell migration: indicators of inefficient lamellipodia adhesion and compartments of actin filament reorganization.</a> Exp. Cell. Res. 302, 83-95 (2005).</li><li>Hotulainen, P., Lappalainen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+fibers+are+generated+by+two+distinct+actin+assembly+mechanisms+in+motile+cells.">Stress fibers are generated by two distinct actin assembly mechanisms in motile cells.</a> J. Cell. Biol. 173, 383-394 (2006).</li><li>Pellegrin, S., Mellor, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+stress+fibres.">Actin stress fibres.</a> J. Cell. Sci. 120, 3491-3499 (2007).</li><li>Ballestrem, C., Wehrle-Haller, B., Imhof, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+dynamics+in+living+mammalian+cells.">Actin dynamics in living mammalian cells.</a> J. Cell. Sci. 111, 1649-1658 (1998).</li><li>Helmke, B. P., Rosen, A. B., Davies, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+mechanical+strain+of+an+endogenous+cytoskeletal+network+in+living+endothelial+cells.">Mapping mechanical strain of an endogenous cytoskeletal network in living endothelial cells.</a> Biophys. J. 84, 2691-2699 (2003).</li><li>Birukova, A. A., Smurova, K., Birukov, K. G., Kaibuchi, K., Garcia, J. G., Verin, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Rho+GTPases+in+thrombin-induced+lung+vascular+endothelial+cells+barrier+dysfunction.">Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction.</a> Microvasc. Res. 67, 64-77 (2004).</li><li>van Nieuw Amerongen, G. P., van Delft, S., Vermeer, M. A., Collard, J. G., van Hinsbergh, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+RhoA+by+thrombin+in+endothelial+hyperpermeability:+role+of+Rho+kinase+and+protein+tyrosine+kinases.">Activation of RhoA by thrombin in endothelial hyperpermeability: role of Rho kinase and protein tyrosine kinases.</a> Circ. Res. 87, 335-340 (2000).</li><li>Moy, A. B., Blackwell, K., Kamath, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+histamine+and+thrombin+on+endothelial+barrier+function+through+actin-myosin+tension.">Differential effects of histamine and thrombin on endothelial barrier function through actin-myosin tension.</a> Am J Physiol Heart Circ Physiol. 282, H21-H29 (2002).</li><li>Wittman, T., Littlefield, R., Waterman-Storer, C., Goldman, R. D., Spector, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+speckle+microscopy+of+cytoskeletal+dynamics+in+living+cells.">Fluorescent speckle microscopy of cytoskeletal dynamics in living cells.</a> Live Cell Imaging: A Laboratory Manual. , 184-204 (2005).</li><li>Rabut, G., Ellenberg, J., Goldman, R. D., Spector, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobleaching+techniques+to+study+mobility+and+molecular+dynamics+of+proteins+in+live+cells:+FRAP,+iFRAP,+and+FLIP.">Photobleaching techniques to study mobility and molecular dynamics of proteins in live cells: FRAP, iFRAP, and FLIP.</a> Live Cell Imaging: A Laboratory Manual. , 101-126 (2005).</li></ol>

利害の衝突は宣言されません。

生内皮細胞におけるGFP -アクチンのイメージングは​​、炎症性刺激に反応してアクチン細胞骨格のダイナミクスの詳細な分析が可能になります。また、このメソッドは、せん断応力11のように物理的な力に反応して細胞骨格のリモデリングを示すこれまでの知見に基づいて構築すると便利な場合があります。さらに、この方法は、マイグレーション、有糸分裂、細胞間接合部と接合部の成熟の形成、及びバリア機能の維持など、さまざまな内皮細胞の活動へのアクチン細胞骨格の動態の寄与の詳細な評価が可能になります。 示されたデータでは、内皮のアクチン細胞骨格の挙動は、トロンビンによる治療前後で観察することができます。すべての内皮細胞の縁に沿って地元の葉状仮足を形成し、非融合と合流両方の細胞の単層で時間をかけて回帰が観察された。トロンビンによる治療は一時的に葉状仮足の形を中断ationと離職。トロンビンはまた、トロンビンは内皮細胞12-14にアクチンストレスファイバーの形成と増加求心張力発生を引き起こすことが以前の報告と一致して、細胞がわずかに縮小していました。しかし、このような生細胞イメージング研究から、ストレスファイバーの起源は、今決定することができます。 HUVECにおいては、ストレスファイバーのほとんどは、細胞の周囲に発信し、細胞の8,9の移行に横アーク繊維に似ている。固定された細胞を使用する場合に比べてこの方法のもう一つの強みは、ストレスファイバーの数は、実験群の間で選択バイアスを排除し、トロンビンの治療の前と後の個々の細胞で定量化できることです。 このプロトコルでは、セルのエッジとアクチンストレスファイバーの動的な動きを評価する。内皮細胞でのアクチンの単量体のダイナミクスを理解するために、このような蛍光退色後回復（FRAP）や蛍光などのより高度な技術がマイクロスペックルコピー（FSM）は、16、15を適用することができます。微小血管内皮細胞は微小血管のバリア機能のより良いモデルを表すことができるので、さらに、効果的に微小血管内皮細胞にGFP -アクチンを発現するようにトランスフェクションプロトコールの最適化は論理的な将来の方向を表します。 要約すると、GFP -アクチンを発現している生内皮細胞のイメージングは​​、内皮細胞のアクチン細胞骨格は、刺激の様々なタイプに応答する方法を決定する強力なツールを表します。しっかりと密内皮単層を用いた研究は、このようなアクチンに富んだ葉状仮足および内皮バリア機能の横アークストレスファイバーなどの動的構造の役割を決定するのに役立ちます。さらに、GFP -アクチンまたは他の細胞内構造の可視化を可能にする他の融合タンパク質を発現する内皮細胞の生細胞イメージングでは、シグナリングと構造的なメカニズムを理解するために必要な詳細な時空間情報を提供することをDetermineバリアの完全性。

<table border="1"><tbody><tr><td colspan="4"> 1。リンガー5倍証券 </td></tr><tr><td> 化学 </td><td> 会社 </td><td> カタログ番号 </td><td> 量は </td></tr><tr><td>塩化ナトリウム</td><td> EMD </td><td> SX0420 - 3 </td><td> 35グラム</td></tr><tr><td>塩化カリウム</td><td> JTベーカー</td><td> 3040 </td><td> 1.75グラム</td></tr><tr><td>塩化カルシウム</td><td>シグマ</td><td> C - 3881 </td><td> 1.47グラム</td></tr><tr><td>硫酸マグネシウム</td><td>シグマ</td><td> M - 9397 </td><td> 1.44グラム</td></tr><tr><td>滅菌ろ過された水</td><td> N / </td><td> N / </td><td> 1 Lへ移動</td></tr><tr><td colspan="4"> 4℃オートクレーブボトルや店舗に滅菌フィルターは、° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 2。 MOPSバッファー </td></tr><tr><td> 化学 </td><td>会社 </td><td> カタログ番号 </td><td> 量は </td></tr><tr><td> MOPS </td><td>シグマ</td><td> M3183 </td><td> 125.6グラム</td></tr><tr><td>滅菌ろ過された水</td><td> N / </td><td> N / </td><td> 1 Lへ移動</td></tr><tr><td colspan="4"> 4℃オートクレーブボトルや店舗に滅菌フィルターは、° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 3。アルブミン生理食塩水（APSS） </td></tr><tr><td> 化学 </td><td> 会社 </td><td> カタログ番号 </td><td> 量は </td></tr><tr><td>リンガー株式（5倍） </td><td> N / </td><td> N / </td><td> 200 mLの</td></tr><tr><td> MOPS緩衝液</td><td> N / </td><td> N / </td><td> 5 mLの</td></tr><tr><td>リン酸ナトリウム</td><td>シグマ</td><td> S - 9638 </td><td> 0.168グラム</td></tr><tr><td>ピルビン酸ナトリウム&lt;/ TD&gt; <td>シグマ</td><td> P5280 </td><td> 0.22グラム</td></tr><tr><td> EDTAのナトリウム塩</td><td>シグマ</td><td> ED2SS </td><td> 0.0074グラム</td></tr><tr><td>グルコース</td><td>シグマ</td><td> G7528 </td><td> 0.901グラム</td></tr><tr><td>アルブミン、ウシ</td><td> USB </td><td> 10856 </td><td> 10グラム</td></tr><tr><td>滅菌ろ過された水</td><td> N / </td><td> N / </td><td> 1 Lへ移動</td></tr><tr><td colspan="4"> 37 7.4にpHを調整° C、4℃オートクレーブボトルや店舗にして滅菌フィルター℃に</td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 4。 0.9％生理食塩水 </td></tr><tr><td> 化学 </td><td> 会社 </td><td> カタログ番号 </td><td> 量は </td></tr><tr><td>塩化ナトリウム</td><td> EMD </td><td> SX0420 - 3 </td><td> 9グラム</td></tr><tr><td>滅菌ろ過された水</td><td> N / </td><td> N / </td><td> 1 Lへ移動</td></tr><tr><td colspan="4"> 4℃オートクレーブボトルや店舗に滅菌フィルターは、° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 5。 1.5パーセントゼラチン溶液 </td></tr><tr><td>ブタの皮からゼラチン</td><td>シグマ</td><td> G2500 </td><td> 15グラム</td></tr><tr><td> 0.9％生理食塩水</td><td> N / </td><td> N / </td><td> 1 Lへ移動</td></tr><tr><td colspan="4"> ° C十分にゼラチン溶解するために37にソリューションを温める。一方、4℃オートクレーブ滅菌ボトルとストアにまだ温かい、滅菌フィルター° C </td></tr></tbody></table>

study of the actin cytoskeleton in live endothelial cells expressing gfp-actin

微小血管内皮細胞は、流体と溶質の選択的透過性障壁として重要な役割を果たしている。内皮細胞間の接着剤接合は、内皮の透過性を調節し、そして多くの研究では、接合部の整合性1-5を決定するために、アクチン細胞骨格の重要な貢献を示している。皮質アクチンのベルトは、安定した接合部1、2、4、5の維持に重要であると考えられている。対照的に、アクチンストレスファイバーは接合2-5を弱める内皮細胞内の求心緊張を生成すると考えられている。ずっとこの理論のは内皮細胞が血管内皮透過性を高めるために知られている炎症性メディエーターで処理し、細胞を固定し、顕微鏡観察のためにF -アクチンのラベル付けされた研究に基づいている。に固定された細胞の画像のみスナップショットを提供するため、しかし、これらの研究は、アクチン細胞骨格の役割を非常に限られた理解を提供するアクチンの構造5のダイナミクスに関する情報がないと時間。 生きた細胞のイメージングは​​、内皮障壁の完全性を決定するメカニズムの研究にアクチン細胞骨格の動的な性質に取り込むことができます。この方法の大きな利点は、内皮細胞でのアクチンの構造上の様々な炎症性刺激の影響は、固定標本を観察するときに発生する可能性のある潜在的なバイアスを除去する、治療前と後の生細胞の同じセットで評価することができるということです。ヒト臍帯静脈内皮細胞（HUVEC）をプラスミドGFP -β-アクチンをトランスフェクションし、ガラスのカバースリップ上でコンフルエンスまで増殖されています。コンフルエントHUVECにおけるGFP -アクチンの時間経過の画像は、内皮障壁の完全性の時間依存性の変化を誘導する炎症性メディエーターを添加する前と後にキャプチャされます。これらの研究は、内皮に貢献するアクチン細胞骨格の変化の流体シーケンスの視覚的な観察が可能lのバリアの破壊と修復。 我々の結果は一貫して内皮細胞の局所、アクチン豊富な葉状仮足の形成と売上高を示しています。アクチンストレスファイバーの形成と動きも観察することができます。炎症性刺激による治療の前と後のローカル葉状仮足の形成やターンオーバーの頻度の分析は、カイモグラフ解析によって文書化することができます。これらの研究は、内皮障壁の完全性の維持のための重要な以前に正体不明の分子機構を発見するために使用することができる内皮細胞におけるアクチン細胞骨格の動的な性質に関する重要な情報を提供しています。

Watch this Scientific Journal Video about Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin at JoVE.com

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

GFP -アクチンを発現している生内皮細胞の顕微鏡イメージングは​​、細胞骨格構造の動的変化の特性評価が可能になります。固定標本​​を使用する技法とは異なり、この方法は前と同じ細胞内のアクチン細胞骨格の時間的変化の詳細な評価、中、および様々な物理的、薬理学的、または炎症性刺激の後にしています。

GFP -アクチンを発現するライブ内皮細胞におけるアクチン細胞骨格の研究

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

study-actin-cytoskeleton-live-endothelial-cells-expressing-gfp

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

Unit 1

The Cytoskeleton I: Actin and Microfilaments

SCIENTISTS IN ACTION

27.0K ビュー.  Louisiana State University Health Sciences Center. GFP -アクチンを発現している生内皮細胞の顕微鏡イメージングは​​、細胞骨格構造の動的変化の特性評価が可能になります。固定標本​​を使用する技法とは異なり、この方法は前と同じ細胞内のアクチン細胞骨格の時間的変化の詳細な評価、中、および様々な物理的、薬理学的、または炎症性刺激の後にしています。

ビデオ: Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

トカゲの運動の速度と方向の視覚感度のテスト

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

生物学

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

蛍光を経由してF -アクチンダイナミクスの生細胞イメージングでは、顕微鏡のスペックル（FSM）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

細胞運動やゼブラフィッシュ胚における個々の神経細胞と神経堤細胞のアクチン細胞骨格のライブイメージング

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

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 ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

凍結保存サンプルの回復のための日曜大工のデバイスが誤って低温貯蔵タンクに落とし

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

アポトーシス細胞のクリアランスのイメージング中に生きる<em&gt;ショウジョウバエ</em&gt;胚

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

蛍光タンパク質を発現する生きたマウスでのイメージング細胞内の構造物の生体顕微鏡

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

のための内皮細胞チューブ形成アッセイ<em&gt;体外</em&gt;血管新生に関する研究

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

ネズミ弁内皮細胞の単離

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

リビングヒト臍帯静脈内皮細胞における高解像度タイムラプスイメージングと微小管ダイナミクスの自動解析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Methods Article

GFP -アクチンを発現するライブ内皮細胞におけるアクチン細胞骨格の研究