ビンキュリン- GFPプラスミドは、歯科および頭蓋顔面研究所の国立研究所のケネス山田、国立衛生研究所から贈られた、プラスミドベセスダ、メリーランド州、およびアクチン- MRFPはマイケルソン、フロリダ州立大学、タラハシー、フロリダ州から寄贈されたもの。 この作品は、Andreea TracheにNSFのキャリア賞＃0747334とAHA -ナショナルSDG＃0835205Nによってサポートされていました。

1。統合顕微鏡システムの概要これらの研究に使用される顕微鏡システムは、詳細1に記載されている。簡単に言えば、TIRFアタッチメント（オリンパス、センターバレー、ペンシルバニア州）と反転オリンパスIX - 81顕微鏡は、CSU - 22横河スキャニングヘッド（横河電機株式会社、日本）と組み合わされる。ビオスコープSZ原子間力顕微鏡（Veeco社インスツルメンツ社、サンタバーバラ、カリフォルニア州）は、光学倒立顕微鏡の上部に取り付けられており、全体のシステムは研究用光学テーブルの上（ニューポート、アーバイン、カリフォルニア州）に配置されます。 回転ディスクスキャナー、外付けのフィルターホイールとそのEMCCDカメラ（ローパーサイエンティフィック- Photometrics、ツーソン、アリゾナ州）から構成される共焦点アセンブリは厳密に振動減衰のために必要なシリコンパッド上に配置アルミベースプレートと接続​​されています。共焦点スキャナは、回転ディスクスキャナからの振動がAFMの測定に影響を与えるしませんようにAFM測定、中に顕微鏡から切り離すことができる踏み襟付きのフランジを介して顕微鏡に接続します。アルゴンクリプトンレーザー（スペクトラフィジックス、マウンテンビュー、カリフォルニア州）はそれぞれ、全反射と共焦点イメージングの両方のモードの励起源として使用され、全反射の添付ファイルまたは走査ヘッドにレーザーを提供する2本の光ファイバで交互に結合することができます。 。 2台のコンピュータがシステムを制御します。Slidebookソフトウェアは、光学イメージングを制御し、ナノスコープのソフトウェアは、AFMを制御します。 2。細胞の文化特定のタンパク質を標的とGFPコンストラクトを発現している生きている細胞は、24時間前に機械的刺激実験にグラスボトム細胞の培養皿に配置する必要があります。実験日にフェノールレッドを含まない培地で細胞培養の培地を交換してください。細胞培養ディッシュは、磁気襟とAFMステージに搭載されている。 3。 AFMチップの調製と顕微鏡のセットアップ 2ミクロンでカスタマイズAFMチップは、ガラスビーズをDPBSで5回洗浄し、アビジン（1 mg / ml）を、5分間インキュベートし、DPBS 5回で再び洗浄し、1 mg / mLのbiotinilatedで5分間インキュベートされますbiotinilatedフィブロネクチン。先端には、エンド2でさらに5回洗浄し、AFMスキャナ上にマウントし、保護シリコンスカートは、ホルダーの周囲に配置されますされます。視野の中心にAFMの先端を揃えるために、ビデオ視聴のための顕微鏡を設定します。カンチレバーの一番最後にレーザービームを配置することにより、光てこを合わせ、単一細胞イメージングのための適切な高倍率にする目標を変更してください。先端のばね定数は、実験開始前に決定する必要があります。このパラメータは、ナノスコープのソフトウェアで利用可能な熱調整法を用いて測定されます。蛍光イメージングを可能にするためにビデオカメラからEMCCDカメラに切り替えます。その後、セルを選択し、その特定の細胞と接触AFMの先端をもたらす。 20分が細胞表面との接触AFMの時点で形成するために強力な接着斑のために必要な時間を、待機した後、機械的な刺激の手続きが開始されます。 4。ライブセルの機械的刺激 3月4日全反射イメージングは、接着斑の可視化と定量化のための選択の方法となり、回転ディスク共焦点イメージング5は、細胞体全体にアクチン細胞骨格を可視化するために使用されます。 AFM 6は流体下での接触モードのイメージングに使用されます。 セルの全反射や共焦点画像は、機械的な刺激の手順を開始する前に取得されます。機械的な刺激は、一定期間（1〜1.5時間）以上のすべての3〜5分離散的なステップで上方に移動される官能化されたAFMのプローブによって誘導されます。細胞の反応性応答は1秒の単位時間を表す画像の各ライン、高さのイメージとして、ナノスコープのソフトウェアで連続して記録されます。 AFMデータの取得中、オペレータは、常時AFMチップのZ位置は、次のプルダウンのための適切なタイミングを決定するために監視します。 各機械的な刺激の後にそれが継続的にAFMのデータを取得しながら、Slidebookソフトウェアにおけるセルの全反射や共焦点画像を取得することが可能です。光学画像は、アクチン細胞骨格と接着斑の再編成を示してムービーを作成するためにオフラインで処理することができます。また、各画像は、セル面積、特定のタンパク質の領域を決定する、と一般的な細胞形態を分析するために定量的に処理することができます。 実験の終わりにAFMの先端が細胞から回収され、AFMチップの感度は、ナノスコープのソフトウェアで測定されます。 実験は、非常にノイズに敏感です！ EX中に触れてどんな話したり、顕微鏡perimentは避けてください。 AFM測定でノイズを最小限に抑えるために、回転するディスク共焦点は、顕微鏡からデ結合してください。細胞表面と探針の間に強力な接着斑を形成するために待っている時間は、使用する細胞の種類、チップ上の行列のコーティングによって異なる場合があります。 5。代表的な結果<img src="/files/ftp_upload/2072/2072fig1.jpg" alt="図1" /> 図1。アクチン- MRFPとビンキュリン- GFPをトランスフェ同じ血管平滑筋細胞（VSMC）は次式で画像化した。頂端細胞表面での（A）AFM、（B）基底細胞の表面で全反射、および（C）回転ディスク共焦点顕微鏡細胞体全体に。共焦点画像は3 - Dスタックの最大投影として示されています。 AFMとTIRFや回転するディスク共焦点画像間の良好なオーバーラップは（D）及び（E）、それぞれに示されています。画像サイズは64 × 50ミクロンです。 <img src="/files/ftp_upload/2072/2072fig2.jpg" alt="図2" /> 図2。アクチン- MRFPでトランスフェクトしたVSMCは、機械的にAFMによって刺激され、回転するディスク共焦点顕微鏡で画像化した矢印はそのアクチン繊維を示す：（a）は厚く/重合、（b）の解重合/消える、または（c）により、一緒に結束アクチンの再構築へ。白い点線は、セルの上にAFMチップを表します。画像サイズポスター91 x 91ミクロン。

<ol>
	<li>Trache, A., Lim, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+microscopy+for+real-time+imaging+of+mechanotransduction+studies+in+live+cells.">Integrated microscopy for real-time imaging of mechanotransduction studies in live cells.</a> J Biomed Opt. 14, 034024-03 (2009).</li><li>Trache, A., Meininger, G. A., Coico, R., Kowalik, T., Quarles, J. M., Stevenson, B., Taylor, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+Force+Microscopy.">Atomic Force Microscopy.</a> Current Protocols in Microbiology. , 2.1-2.17 (2008).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+imaging+of+surface+fluorescence+with+very+high+aperture+microscope+objectives.">Selective imaging of surface fluorescence with very high aperture microscope objectives.</a> J Biomed Opt. 6, 6-13 (2001).</li><li>Trache, A., Meininger, G. A., Coico, R., Kowalik, T., Quarles, J. M., Stevenson, B., Taylor, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+Internal+Reflection+Fluorescence+Microscopy.">Total Internal Reflection Fluorescence Microscopy.</a> Current Protocols in Microbiology. , 2.1-2.22 (2008).</li><li>Inoue, S., Inoue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-view+high-speed+confocal+scanner:+the+CSU-10.">Direct-view high-speed confocal scanner: the CSU-10.</a> Methods Cell Biol. 70, 87-127 (2002).</li><li>Binnig, G., Quate, C. F., Gerber, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscope.">Atomic force microscope.</a> Phys Rev Lett. 56, 930-933 (1986).</li><li>Stamenovi&#263;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+mechanics+in+airway+smooth+muscle+cells.">Cytoskeletal mechanics in airway smooth muscle cells.</a> Respir Physiol Neurobiol. 163, 25-32 (2008).</li><li>Gunst, S. J., Tang, D. D., Opazo Saez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+remodeling+of+the+airway+smooth+muscle+cell:+a+mechanism+for+adaptation+to+mechanical+forces+in+the+lung.">Cytoskeletal remodeling of the airway smooth muscle cell: a mechanism for adaptation to mechanical forces in the lung.</a> Respir Physiol Neurobiol. 137, 151-168 (2003).</li><li>Worth, N. F., Rolfe, B. E., Song, J., Campbell, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+smooth+muscle+cell+phenotypic+modulation+in+culture+is+associated+with+reorganization+of+contractile+and+cytoskeletal+proteins.">Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganization of contractile and cytoskeletal proteins.</a> Cell Motility and the Cytoskeleton. 49, 130-145 (2001).</li><li>Romer, H. R., Birukov, K. G., Garcia, J. G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+Adhesions:+paradigm+for+a+signaling+nexus.">Focal Adhesions: paradigm for a signaling nexus.</a> Circulation Research. 98, 606-616 (2006).</li><li>Opazo Saez, A., Zhang, W., Wu, Y., Turner, C. E., Tang, D. D., Gunst, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tension+development+during+contractile+stimulation+of+smooth+muscle+requires+recruitment+of+paxillin+and+vinculin+to+the+membrane.">Tension development during contractile stimulation of smooth muscle requires recruitment of paxillin and vinculin to the membrane.</a> Am J Physiol Cell Physiol. 286, C433-C447 (2004).</li><li>Alenghat, F. J., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction:+all+signals+point+to+cytoskeleton,+matrix+and+integrins.">Mechanotransduction: all signals point to cytoskeleton, matrix and integrins.</a> Sciences STKE. 119, pe6-pe6 (2002).</li><li>Zamir, E., Geiger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+complexity+and+dynamics+of+cell-matrix+adhesions.">Molecular complexity and dynamics of cell-matrix adhesions.</a> J Cell Sci. 114, 3583-3590 (2001).</li><li>Geiger, B., Bershdsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+mechanosensory+function+of+focal+contacts.">Assembly and mechanosensory function of focal contacts.</a> Curr Opin Cell Biol. 13, 584-592 (2001).</li><li>Sastry, S. K., Burridge, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+Adhesions:+a+nexus+for+intracellular+signaling+and+cytoskeletal+dynamics.">Focal Adhesions: a nexus for intracellular signaling and cytoskeletal dynamics.</a> Exp Cell Res. 261, 25-36 (2000).</li><li>Orr, A. W., Helmke, B. P., Blackman, B. R., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+mechanotransduction.">Mechanisms of mechanotransduction.</a> Dev Cell. 10, 11-20 (2006).</li></ol>

利害の衝突は宣言されません。

細胞骨格7-10と接着斑11-16細胞が移動または機械的な力17に対応するように、組み立て分散とめくる動的な構造です。彼らは、細胞増殖、生存、および遺伝子発現の調節において重要な機能を持っています。彼らの特定の分布と動態を知ることは、細胞がマトリックスで、自分自身との間の通信方法のより良い理解を提供します。 この新しい顕微鏡...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>AFM tip</td>
<td>&nbsp;</td>
<td>Novascan Technologies</td>
<td>&nbsp;</td>
<td>2 micron Borosilicate bead, 0.01N/m, Biotin Surface</td>
</tr>
<tr>
<td>avidin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>A9275</td>
<td>1mg/mL in DPBS</td>
</tr>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>F4759</td>
<td>1 mg/mL</td>
</tr>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21600-051</td>
<td>&nbsp;</td>
<tr>
<td>Amaxa transfection kit for Smooth Muscle Cells</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>VPI-1004</td>
<td>&nbsp;</td>
<tr>
<td>Mattek glass bottom dishes</td>
<td>&nbsp;</td>
<td>MatTek Corporation</td>
<td>P60G-1.5-30-F</td>
<td>Sterile 60mm Falcon dishes with glass bottom coverslip no. 1.5</td>
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live cell response to mechanical stimulation studied by integrated optical and atomic force microscopy

高い空間分解能と時間分解能を持つサブ細胞レベルでの細胞の挙動を調査することができる新技術、生きている細胞は機械的な力を感知するメカニズムを理解するために、およびそれらがどのように対応し、その環境に適応することは開発されました。このように、原子間力顕微鏡（AFM）は、全内部反射蛍光（全反射）顕微鏡と高速回転ディスク（FSD）共焦点顕微鏡と統合されました。統合されたシステムは、リアルタイムでの機械的刺激への細胞応答の直接光イメージングを可能にする、任意の付着生細胞における分子動力学研究の広い範囲にわたって広く適用可能である。アクチンフィラメントと接着斑の大幅な再編成が原因で、細胞体全体の細胞構造に変化を誘導することが頂端細胞の表面での局所力学刺激することが示された。これらの革新的な技術は、機械的な力に反応して生細胞の再構築とダイナミクスを理解するための新たな情報を提供します。機械的な刺激にショーの生細胞応答が記載されている詳細なプロトコールと代表的なデータセット。

Watch this Scientific Journal Video about Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy at JoVE.com

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

本論文では、培養中の生細胞の機械的刺激のための統合された原子間力光学イメージング顕微鏡の操作で、リーダーに指示することを目指しています。ステップバイステップのプロトコルが表示されます。機械的な刺激に生細胞の応答を示す代表的なデータセットが表示されます。

集積光と原子間力顕微鏡による研究機械的刺激に対する細胞応答を生きる

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

live-cell-response-to-mechanical-stimulation-studied-integrated

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.2K ビュー.  Texas A&M Health Science Center. 本論文では、培養中の生細胞の機械的刺激のための統合された原子間力光学イメージング顕微鏡の操作で、リーダーに指示することを目指しています。ステップバイステップのプロトコルが表示されます。機械的な刺激に生細胞の応答を示す代表的なデータセットが表示されます。

ビデオ: Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

位相コントラストおよび微分干渉コントラスト（DIC）顕微鏡

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

生物学

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

原子間力顕微鏡を用いた肺組織のマイクロメカニカル特性

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

軟骨アガロースゲルの機械的刺激

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

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

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

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

単一のビリオン原子間力顕微鏡と超解像蛍光イメージングのサンプル調製

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

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

の剛性を測定します<em&gt; ex vivoで</em原子間力顕微鏡を使用&gt;マウス大動脈

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

磁気ビーズを使用した機械的張力に応答して細胞表面接着リフォームの分析

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

インスリン刺激に対するグルコース取り込み測定と応答<em&gt;インビトロ</em&gt;培養ヒト初代筋管

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

キイロタマホコリカビ急性の機械的刺激に対する応答の評価

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

突起力顕微鏡: 細胞突起が開発した力を定量化する方法

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...

集積光と原子間力顕微鏡による研究機械的刺激に対する細胞応答を生きる

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