我々は、NIBIB、NIHの学内研究プログラムで研究支援を認める。

サンプルセルアセンブリとトルク<ol><li>我々は、2つのウィンドウのアセンブリを一緒に置くことによって、試料セルの建設を開始。 ウィンドウホルダーにウィンドウのガスケットを置きます。ライナーの開口部がホルダーのキー溝の反対側になるようにウィンドウのホルダー内のウィンドウのライナーを（または、それが時々窓のクッションと呼ばれる）に置きます。少し傾けて、ウィンドウのホルダーのキーの方法でウィンドウ上のスクライブマークを合わせてホルダーにウィンドウを配置します。ウィンドウの両側に非常に端に軽く下に押します。 </li><li>適切なシールとサンプルセルの正確なトルクを確保するために、我々軽くコートSpinkote潤滑剤付き住宅のガスケットとねじリング。親指と人​​差し指の間にSpinkoteの非常に少量を広げる。 Spinkoteの薄い、目に見えない薄膜でコーティングスクリューリングスレッドを。同様に、コートハウジングガスケットを。目に見える潤滑剤を拭き取ってください。 </li><li>住宅のキーでキー溝を合わせてセルのハウジング内に、ウィンドウを手前上に向けて、つのウィンドウのアセンブリをスライドさせて、試料セルのアセンブリを開始します。住宅の鍵で目玉のキー溝を合わせ、静かに細胞内でウィンドウアセンブリの上に落としてください。 </li><li>セルのハウジング内に中心をプッシュするためのツールの任意の並べ替えを使用しないでください。これは、実験中にリークが発生CPに永久的な損傷を引き起こす可能性があります。ウィンドウは、あなたから離れて、中心に向かって直面しているように、2番目のウィンドウアッセンブリを回し、住宅の鍵とキー溝を合わせ、センターピースの上にそれを下にスライドさせます。単語&quot;アウト&quot;が表示されるように、その後、上部のネジリングをハウ​​ジングのガスケットを置きます。あなたの指とアライメントツールを使用して、手はスクリューリングを締め付けます。 CPは、住宅のバレルに簡単にスライドしない場合は、最初にそれを合わせ、その後、それの上に別のウィンドウのアセンブリを配置します。ウィンドウアセンブリの穏やかな下方圧力を適用することによって、同時にセルのハウジング内に両方をスライドさせます。このように、我々は、CPで直接押すことは避けてください。 今は埃、糸くずや指紋のセルの内部を見るのに良い時期です。また、CPと窓の間に空気が残っていることを示すニュートンリングに気づくでしょう。これらは、トルクを適用した後に消えます。 </li><li>ネジのリング上と目に見える&quot;アウト&quot;という言葉で、トルクのスタンドのセルトルクコレット内部試料セルのすべての方法を下にして置かない。トルクのスタンドのハンドルを一定の圧力を適用することによって所定の位置にセルを保持する。つの連続した​​動きで、ポンドで120から140の間にネジリングを締め付けます。調整可能なマイクロメータのトルクレンチを使用している場合は、/ポンドで120から140の間にそれを設定し、設定トルクに達したことを示すレンチ&quot;クリック&quot;するまで、ねじリングを締め付けます。トルクのスタンドのハンドルを解放し、慎重に窓を触れることなく、試料セルを削除します。 </li></ol>サンプルセルのロードとシーリング<ol start="6"><li>沈降速度の実験のために、我々は日常的に200ulのピペットを使用して12ミリメートルセンターピースの各部門に400ulのボリュームをロードする。トップと目に見えるネジのリング上で&quot;アウト&quot;という言葉で読み込む穴に試料セルを配置します。ロードの穴を通して左中心の部門にピペットチップを半押しスライドさせ、徐々に標準溶液の200ulを分注する。 400ulを合計して一回繰り返します。同じピペットチップと同じテクニックを使用して、慎重にゆっくりと試料溶液の400ulとRIGHT目玉分野を埋める。 150から180 ULのボリュームは、沈降平衡実験に使用されますが、同じ注意とテクニックに従ってください。 それは、できる限り緊密にリファレンスとサンプルソリューションのボリュームと一致することが不可欠です。ローディングバブルを回避し、両方のソリューションをできるだけ均等にボリュームを維持するのにすべての援助のために同一のピペットチップを使用して、徐々にソリューションをロードし、調剤、しっかりとピペットにピペットチップの取り付け。 </li><li>彼らはセンターピースとアルミ製のハウジングとの間に配置されていることを確認し、各荷重の穴にぴったりと2つの新しい赤い住宅のプラグのガスケットを配置することにより、セルをシール。住宅のプラグをそれぞれカバー。手には小型のマイナスドライバでネジを締めます。 </li></ol>ローターのサンプルセル配置<ol start="8"><li>ローターをロードする前に、対抗勢力がそのサンプルセルのリーク場合に対向する位置に試料セルを超えない0.5未満グラム軽量であることを確認してください。ゼロ残高から始めます。最初のCBは逆の位置になるサンプルセルの重量を量る。サンプルセルの重量を減算する天びんは、その後、CBの重量を量る。カウンターバランスの取付穴からの重量を加算または減算。重みは上からはみ出さないよう確認してください。対抗勢力の最終重量は0.5グラム内に反対の試料セルに比べて軽量であることを保証するために重さを量り直す。 </li><li> 4場所のローターを使用して、白い矢印が表示されるように位置4にカウンターバランスを置いて、tに指し遠心力の彼の方向。それがダウンしてローター内部のすべての方法であることを確認してください。対抗勢力はまだ整列することができる場所にしっかりとあるが、まで、慎重にちょうど白い矢印の向かいにカウンターバランスの上に見られる一連のネジを、締め。ハウジングとバランスのとれたサンプルセルの位置をローターの中心と目に見えるネジリング上の単語&quot;out&quot;を直面しているプラ​​グ。 </li><li>ローターの下から見ると、整列ツールを使用して、カウンター上のスクライブマークと内側のスクライブマークを（ローターの中心に最も近いマーク）に合わせます。場所に並んでカウンターバランスをロックする止めネジを締めます。同じように、ロータ上の位置2に内部のスクライブマークのついた試料セルのスクライブマークを合わせます。 </li></ol>詳細については参照してください<a href="https://sedfitsedphat.nibib.nih.gov/default.aspx">https://sedfitsedphat.nibib.nih.gov/default.aspx</a>

<ol>
	<li>Brown, P. H., Balbo, A., Schuck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+protein-protein+interactions+by+sedimentation+velocity+analytical+ultracentrifugation.">Characterizing protein-protein interactions by sedimentation velocity analytical ultracentrifugation.</a> Curr Protoc Immunol. Chapter 18, Unit 18.15-Unit 18.15 (2008).</li><li>Balbo, A., Brown, P. H., Braswell, E. H., Schuck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+protein-protein+interactions+by+equilibrium+sedimentation.">Measuring protein-protein interactions by equilibrium sedimentation.</a> Curr Protoc Immunol. Chapter 18, Unit 18.8-Unit 18.8 (2007).</li><li>. Home - Workshops [Internet] Available from: <a target="_blank" href="https://sedfitsedphat.nibib.nih.gov/workshop/default.aspx">https://sedfitsedphat.nibib.nih.gov/workshop/default.aspx</a></li></ol>

ローターの適切なセルアセンブリ、サンプルの挿入、およびセルの配置は、高品質のAUCの実験のための非常に重要です。これは、タンパク質の相互作用を研究し、トレース集計量を決定するなどの高分子の沈降プロファイルの取得したデータを、、の非常に詳細な分析を必要とする実験のために、特に、真です。多くの場合、実験の品質は、AUCの研究の結果の正確さと精度のために制限しています。 3 - 実用的な設計とAUCの実験のさらなる実行の詳細については、参考文献1を参照してください。実用的なセットアップをカバーするワークショップ、理論、およびAUCの実験のデータ解析は、国立衛生研究所の研究室[4] 1年に2回程度開催されている。

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>4-place Ti analytical rotor</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>361964</td>
<td>&nbsp;</td>
<tr>
<td>Torque stand assembly</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>361318</td>
<td>&nbsp;</td>
<tr>
<td>Cell alignment tool</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>362340</td>
<td>&nbsp;</td>
<tr>
<td>Spinkote Lubricant</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>306812</td>
<td>&nbsp;</td>
<tr>
<td>Counterbalance</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>360219</td>
<td>&nbsp;</td>
<tr>
<td>Cell Housing kit</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>334602</td>
<td>&nbsp;</td>
<tr>
<td>Window gasket</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>327021</td>
<td>&nbsp;</td>
<tr>
<td>Window liner</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>362329</td>
<td>&nbsp;</td>
<tr>
<td>Window, sapphire</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>307177</td>
<td>&nbsp;</td>
<tr>
<td>Window, quartz</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>301730</td>
<td>&nbsp;</td>
<tr>
<td>Window holder</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>305037</td>
<td>&nbsp;</td>
<tr>
<td>12mm double sector Epon centerpiece</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>306493</td>
<td>&nbsp;</td>
<tr>
<td>Cell plug gaskets</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>327022</td>
<td>&nbsp;</td>
<tr>
<td>Cell housing plug	</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>362327</td>
<td>&nbsp;</td>		</tbody>
		</table>
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assembly, loading, and alignment of an analytical ultracentrifuge sample cell

分析用超遠心器（AUC）は、私たちは高速遠心分離中に高分子の沈降プロファイルを記録することができる強力な生物物理学的なツールです。適切に計画して実行すると、AUCの沈降速度や沈降平衡実験では、サイズと形状に関しての蛋白質、サンプルの純度、沈降係数、オリゴマー化状態とタンパク質間相互作用について多くを明らかにすることができます。 この手法は、しかし、技術的な関心の厳格なレベルを必要とします。サンプルセルは2つのウィンドウのアセンブリの間に挟まれたセクタセンターピースを保持する。これらは、/ポンドで約120から140のトルクの圧力で密封されています。速度で回転しながら光検出システムが、各中核部門を通して、同じラジアルパスの正中線でのサンプルとリファレンスバッファの両方をスキャンするように、リファレンスバッファおよびサンプルは、目玉のセクターにロードされ、その後、シールした後、細胞を正確にチタン製ローターに並んでいる最大60、000 rpmまでと非常に高真空下でのだけでなく、重要な、適切な試料セルのアセンブリは、サンプルの細胞成分は非常に高価であり、されており、彼らが実験中に漏れや破損を避けるために最適な作業状態であることを確認するため、適切に世話をする必要があります。少しでも亀裂や傷が遠心分離機の破損につながることができるため、慎重に窓を扱う。目玉と窓の間の接触はできるだけタイトにする必要があります。トルクの圧力が印加された後、すなわち無ニュートンのリングが見えるはずです。ほこり、糸くず、窓やセンターピースのどちらかの傷や油のすべての妥協この接触と非常に簡単にすべて一緒に1つのセクタから別のソリューションの漏洩または中心から漏れに​​つながることができます。貴重なサンプルは、実験中のソリューションの漏出、失われる多くの場合、壊れた窓やセンターピースにつながる細胞内の圧力の不均衡の原因となるだけでなく。さらに、プラグのガスケットとハウジングのプラグは、遠心分離室内の高真空での読み込み穴から目玉の部門から引き出されているソリューションを避けるために、所定の位置にしっかり必要があります。ウィンドウのライナーやガスケットは、壊れた窓、結果として運動を引き起こす可能性のある休憩やクラックのない状態でなければなりません。 このビデオでは、コンポーネントの損傷、完璧なAUCの実験中に、ソリューション漏れや破損を最小限にするためにサンプルセルアセンブリ、トルク、負荷及びローターの配置の私たちの手順をデモンストレーションを行います。

Watch this Scientific Journal Video about Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell at JoVE.com

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

分析用超遠心器（AUC）サンプルセルは、サンプルとリファレンスバッファを保持し、実験中に、最大60,000 RPMまで高真空およびローター速度にさらされています。このビデオでは、AUCの実験のこの非常に重要なコンポーネントの組立、荷重と位置合わせのために必要な細部への厳格な注意を紹介します。

アセンブリ、読み込み、および分析用超遠心器のサンプルセルの整列

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

assembly-loading-alignment-an-analytical-ultracentrifuge-sample

Nanyang Technological University

Research

JoVE Journal

Biology

22.5K ビュー.  Dynamics of Macromolecular Assembly, Laboratory of Bioengineering and Physical Science. 分析用超遠心器（AUC）サンプルセルは、サンプルとリファレンスバッファを保持し、実験中に、最大60,000 RPMまで高真空およびローター速度にさらされています。このビデオでは、AUCの実験のこの非常に重要なコンポーネントの組立、荷重と位置合わせのために必要な細部への厳格な注意を紹介します。

ビデオ: Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

アフリカツメガエルの胚および細胞接着アッセイのアセンブリから主催し、動物極の外植片の解剖

生物学

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

腱の生体外で機械的負荷

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

分別アフリカツメガエルの卵抽出を用いたin vitro核アセンブリで

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging

This paper aims to instruct the reader in the assembly and operation of an infrared near-field microscope for imaging beyond the diffraction limit.  The apertureless near-field microscope is a light scattering-type instrument that provides infrared spectra at circa 20 nm resolution. A complete list of components and a step-by-step protocol for use is provided.  Common errors in assembly and instrument tuning are discussed.  A representative data set that shows the secondary structure of an amyloid fibril is presented.  

The assembly of a nearfield infrared microscope for imaging protein aggregates is described.

アセンブリは、チューニングとタンパク質イメージングのためApertureless近距離赤外線顕微鏡の使用

This paper aims to instruct the reader in the assembly and operation of an infrared near-field microscope for imaging beyond the diffraction limit.  The ...

Cargo Loading onto Kinesin Powered Molecular Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport.
These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles 1. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available.
Building on the classic inverted motility assay 2, the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo 3. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface.
The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA4, quantum dots 5 or a wide variety of antigens via biotinylated antibodies 4-6.

Molecular shuttles consisting of functionalized microtubules gliding on surface-adhered kinesin motor proteins can serve as a nanoscale transport system. Here, the assembly of a typical shuttle system is described.

キネシンパワード分子シャトルバスへのロード貨物

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

三次元蛍光画像から細胞形態の変化を定量分析ツール

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

最適STORM超解像顕微鏡のためのテストサンプル

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

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 ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

ギブソンアセンブリを使用して、ヒト伸長因子1αプロモーターの制御下でFLAGタグ化タンパク質を発現するプラスミドベクターの作製

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

TEM分析のための低細胞数とサンプル調製のための代替的アプローチ

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...