研究は、NIHの助成金EY012031とEY0182175とFMカービー財団によってサポートされていました。

文化の中で分離した細胞を操作するための光ピンセット 光ピンセットの捕捉力は光の運動量（; Ashkinら、1986。Ashkin、1991）から生成されます。これらの力は容易に懸濁液中の細胞をトラップが、それらは表面に付着する細胞を移動することはできません。したがって、細胞がトラップされている時点で培養皿への付着を減らすために、カバースリップは、撥のセルを持つポリ-2 -ヒドロキシエチルメタクリレート（ポリ- HEMA）（シグマケミカル社、セントルイス、MO）、毒性のない化合物で被覆されているプロパティは、以前に内皮細胞上の接着試験（フォークマンとモスコーナ、1978）に採用。ポリ- HEMAが培養皿の半分をカバーしながら、残りの半分は、細胞の成長をサポートしている基質でコーティングされています。当研究室では、サル- 1は培養サンショウウオ網膜細胞の基質として使用されます。サル- 1とポリ- HEMAコートディッシュを製造するための手順を以下に示します。 培養皿の作製<ol><li>酸のクリーンcoverglasses（＃1 VWRサイエンティフィック社、メディア、PA）。このカバーガラスは、レーザーを集束させるために必要な高開口数の目的のために必要なだけ0.1mmのその厚さ、のために選ばれました。 </li><li>細胞の撥領域を作成するには、室温で一晩95％エタノール1mlの20 mgのポリ- HEMA（シグマケミカル社、セントルイス、MO）を溶解する。好ましくは、このソリューションは、準備の1ヶ月以内に使用する必要があります。 </li><li>このようなボンネットのようなほこりのない環境で近くに垂直位置にcoverglassesを支える。 </li><li> 200μlのマイクロピペットのプラスチック製の先端からカバーガラスの上部付近にポリ- HEMAの解決方法の1つまたは2滴を置きます。ドロップは（各50 -100μlの約）カバーガラスの半分に制限する必要があります。急な傾斜は、ポリ- HEMAの薄いにもコーティングを提供し、底にカバーガラスの表面下に液体の流れを保証します。ポリ- HEMAコートしたcoverglassesは、室温で30-60分間フード内で乾燥することができます。 </li><li>エッジに近い微細な先端のペンでカバーガラスのポリHEMA側をマーク。 </li><li> 35mmの培養皿の底に1cmの穴を開けます。 </li><li>ポリ- HEMAコーティング面が皿の内側に向かって手前になるように、Sylgard 184（ダウコーニング社、ミッドランド、MI）と培養皿の底に接着剤ポリ- HEMAコートしたカバーガラスを。ポリ- HEMAコーティングの端が穴の中央を下に実行されることを確認します。料理は、ほこりのない環境で、室温で1〜3日間乾燥することができます。 </li><li>ポリ- HEMAコーティングのエッジに続くダイヤモンドペンでカバーガラスの外側の線に傷を付けない。その後、離れてそれを約2mmに直角に二つの交差する線に傷を付けない。交差点の二点は、参照または顕微鏡のステージ上で培養皿の同じ方向を再現するために使用する基準点として機能します。 </li><li>一晩紫外光の下で料理を滅菌する。 </li><li>網膜細胞膜（気前よくピーターMacLeish、医学のモアハウススクールが提供するに対して発生させたマウス抗サンショウウオの抗体を含む培養皿の細胞付着性半分、コートサル- 1と非ポリHEMA半分を、作成するには、アトランタ、ジョージア州、。）MacLeishら（1983）を参照してください。ポリ- HEMA側上で液体をこぼさないように注意しながら、約100μlの0.1 mg / mlの滅菌ヤギ抗マウスIgG抗体（ベーリンガーマンハイム社、インディアナポリス、インディアナ州）の、と最初に、コートサル- 1側を。三時間のために残し、それを取り外します。一度か二度、次に皿のポリ- HEMA側に液体をこぼさないように注意しながら、100μlのサル- 1上清を置く滅菌リンゲル液と同じ部分を洗う。第一抗体はサル- 1は、既知の濃度で塗布されることが保証されます。 </li><li>サル- 1晩で皿をインキュベートする。 </li><li>サル- 1抗体溶液を除去し、滅菌リンゲル液で領域を洗う。細胞播種直前に皿に2mlの培地をご紹介。 </li></ol>両生類リンゲル溶液、無血清培地両生類、および酵素消化のためのリンゲル液の組成はMacLeishとタウンズ - アンダーソン（1988）とマンデルらに記載されています。 （1993）。 細胞培養の準備このビデオで使用されている細胞が由来した明順応、成人、水生相虎サンショウウオは（Ambystoma tigrinum）、長さが17〜22センチメートルを測定する（チャールズサリバン社、ナッシュビル、テネシー州）、5℃に維持Cを上12時間：12時間明暗サイクル。プロトコルは、動物実験によって承認され、国立衛生研究所からのガイドラインに厳密に従い、ニュージャージー医科歯科大学の委員会（IACUC）を使用していた。網膜細胞培養を調製するための手順は、Nachman - Clewnerとタウンズ - アンダーソン（1996）によって記述され、次のとおりですされています。 <ol><li>首を切ると髄動物。目を削除し、角膜と虹彩をばらばらにする。網膜は、アイカップの中でわずかにデタッチしてください。網膜の下に湾曲した鉗子を置くことによって、網膜をかき出す。レンズはこの段階で網膜に接続されている場合、それは重要ではありません。 </li><li> 40分のためのサンショウウオリンゲル溶液に14 U / mlのパパイン（ワーシントン、フリーホールド、ニュージャージー州）で網膜を消化。 </li><li>使用前に、パパインリンゲル溶液を30分間95％O 2 / 5％CO 2ガスをバブリングしてください。、その後0.22ミクロンフィルター（ミリポア、Carrigtwohill、アイルランド）を通過させることによって滅菌。 </li><li>無菌サンショウウオリンゲル液で3回網膜をすすぎます。 </li><li>リンゲル溶液からレンズをはずして、その後静かに細胞懸濁液を得るためにピペットを退屈さ3ミリメートルと網膜組織をひいて粉にする。 </li></ol>分離されたニューロンのレーザーピンセット操作<ol><li>ステージ上の基準点と皿にマークを合わせて料理を向ける、顕微鏡のステージ上に培養皿を置きます。コンピュータの基準点の座標を保存します。 </li><li>料理のサル- 1とポリ- HEMA半分の両方の上に細胞をプレート。 </li><li>細胞がディッシュの接着側の細胞が抗体を基板に接続するための十分な長さは約20分、のために解決することができます。 </li><li>料理のサル- 1接着側のセルを選択します。 xとyのステージは、細胞に近い位置の座標をマーク。私たちのケースでは、ポイントは選択されたセルの一次樹状突起から約10μmのだ。コンピュータの座標を保存します。 </li><li>皿のポリ- HEMA側の私たちの例では、第2ニューロン、感光体を、選択して、それをトラップするために、レーザーピンセットのツールを使用してください。トラッピングは、パワーレベルの広い範囲で達成することができます。しかし、我々はセルと一緒にトラッピング破片を防ぐのに十分な低さが、メディアを介して細胞を輸送するのに十分である、10％-20％で、レーザーのパワーを設定します。 </li><li>トラップ内のセルを保持しながら、細胞がウェル培養皿の表面上および接続されているすべてのニューロン上になるように段階を下げる。サル- 1側で以前に保存されたx座標およびy座標にセルを持って来るために、コンピュータの制御下にステージを移動します。ステージ移動は、8 -20μmの/ sの速度で設定されていましたが、最高速度は、経験的に決定されるべきである。サル- 1側の宛先ポイントに到着、皿の表面にトラップされた細胞を持ってステージを上げる。セルの配置は、数ミクロンの精度で行うことができます。細胞は、サル- 1基質に付着することができます。 </li><li>ペア形成の前と後のペアの両方の細胞のデジタル画像を得ることが有用な記録を提供します。 </li><li>インキュベーターで細胞のペアを維持する。サンショウウオの細胞の場合は、10時、加湿チャンバーに料理を置く℃に細胞は、ビデオの時間の経過によって監視し、必要な細胞生物学的なテクニックを使用することができます。 </li></ol>

<ol>
	<li>Ashkin, A., Dziedzic, J. M., Bjorkholm, J. E., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+a+Single-Beam+Gradient+Force+Optical+Trap+for+Dielectric+Particles.">Observation of a Single-Beam Gradient Force Optical Trap for Dielectric Particles.</a> Opt. Lett. 11, 288-290 (1986).</li><li>Clarke, R. J., Hoegnason, K., Brimacombe, M., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cone+and+rod+cells+have+different+target+preferences+in+vitro+as+revealed+by+optical+tweezers.">Cone and rod cells have different target preferences in vitro as revealed by optical tweezers.</a> Mol. Vision. 14, 706-720 (2008).</li><li>Folkman, J., Moscona, A. <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+cell+shape+in+growth+control.">Role of cell shape in growth control.</a> Nature. 273, 345-349 (1978).</li><li>Liu, Y., Cheng, D. K., Soneck, G. J., Berns, M. W., Chapman, C. F., Tromberg, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+localized+cell+heating+induced+by+infrared+optical+tweezers.">Evidence of localized cell heating induced by infrared optical tweezers.</a> Biophysical Journal. 68, 2137-2144 (1995).</li><li>MacLeish, P. R., Barnstable, C. J., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+monoclonal+antibody+as+a+substrate+for+mature+neurons+in+vitro.">Use of a monoclonal antibody as a substrate for mature neurons in vitro.</a> Proc. Natl. Acad. Sci. USA. 80, 7014-7018 (1983).</li><li>MacLeish, P. R., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+synapse+formation+among+major+classes+of+adult+salamander+retinal+neurons+in+vitro.">Growth and synapse formation among major classes of adult salamander retinal neurons in vitro.</a> Neuron. 1, 751-760 (1988).</li><li>Mandell, J. W., MacLeish, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Townes-Anderson+E.+Process+outgrowth+and+synaptic+varicosity+formation+by+adult+photoreceptors+in+vitro.">Townes-Anderson E. Process outgrowth and synaptic varicosity formation by adult photoreceptors in vitro.</a> J. Neurosci. 13, 3533-3548 (1993).</li><li>Nachman-Clewner, M., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injury-induced+remodelling+and+regeneration+of+the+ribbon+presynaptic+terminal+in+vitro.">Injury-induced remodelling and regeneration of the ribbon presynaptic terminal in vitro.</a> J. Neurocytol. 25, 597-613 (1996).</li><li>Townes-Anderson, E., St Jules, R. S., Sherry, D. M., Lichtenberger, J., Hassanain, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromanipulation+of+retinal+neurons+by+optical+tweezers.">Micromanipulation of retinal neurons by optical tweezers.</a> Mol. Vis.. 4, (1998).</li></ol>

光は勢いがあり、それがセルを通過する光線が屈折されるとき、力が運動量の方向を変更する必要があります。のため運動量保存の法則から、反対方向の力は、順番に、セルに戻って反応する必要があります。 Ashkin（1991）顕微鏡対物レンズで集光レーザービームによって発生する力がフォーカスの中心に向かってセルを移動することを示した。レーザービームは、力のほんの数ピコニュートンを生成する?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>25mm circle No.1 coverglass</td>
<td>&nbsp;</td>
<td>VWR Scientific Inc., Westchester, PA</td>
<td>48380 080</td>
<td>&nbsp;</td>
<tr>
<td>poly-2-hydroxyethylmethacrylate (poly-HEMA)</td>
<td>&nbsp;</td>
<td>Sigma Chemical Co., St Louis, MO</td>
<td>P-3932</td>
<td>Dissolve in 95% ethanol</td>
</tr>
<tr>
<td>Goat anti-mouse IgG antibody</td>
<td>&nbsp;</td>
<td>Chemicon International, Temecula CA</td>
<td>AP181</td>
<td> 1mg in 1ml, dilute 10x for use</td>
</tr>
<tr>
<td>Sal-1 supernatant containing mouse anti-salamander antibody</td>
<td>&nbsp;</td>
<td>generously provided by Dr. Peter MacLeish</td>
<td>&nbsp;</td>
<td>Dr. Peter MacLeish, Morehouse School of Medicine, Atlanta, GA</td>
</tr>
<tr>
<td>3 mm bore 5ml pyrex disposable pipets</td>
<td>&nbsp;</td>
<td>Corning Inc., Corning NY</td>
<td>7078A-5</td>
<td>&nbsp;</td>
<tr>
<td>Cell culture dishes 35mm x 10mm</td>
<td>&nbsp;</td>
<td>Corning inc., Corning NY</td>
<td>430165</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 silicone elastomer kit</td>
<td>&nbsp;</td>
<td>Dow Corning Corp., Midland MI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Optical tweezers-microtool or laser tweezers</td>
<td>&nbsp;</td>
<td>Cell Robotics Inc., Albuquerque NM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 W continuous wave diode laser of 980nm wavelength</td>
<td>&nbsp;</td>
<td>Cell Robotics Inc., Albuquerque NM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Axiovert 100 inverted light microscope</td>
<td>&nbsp;</td>
<td>Carl Zeiss Inc., Thornwood, NY</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>40x oil immersion plan neofluor objective lens</td>
<td>&nbsp;</td>
<td>Carl Zeiss Inc., Thornwood, NY</td>
<td>&nbsp;</td>
<td>Numerical aperture (N.A. 1.3)</td>
</tr>
<tr>
<td>Black and white CCD camera</td>
<td>&nbsp;</td>
<td>Sony Corporation, Tokyo, Japan</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Computer and joystick with software</td>
<td>&nbsp;</td>
<td>Cell Robotics Inc.</td>
<td>&nbsp;</td>
<td>for controlling a motorized stage</td>
</tr>		</tbody>
		</table>
		</div>

using laser tweezers for manipulating isolated neurons in vitro

この論文とビデオでは、我々はin vitroでの成人網膜神経細胞の再生細胞プロセスのターゲット設定を研究する我々の研究室で使用されるプロトコルについて説明します。網膜細胞培養を調製するための手順は、解剖、網膜の消化と粉砕し、特にレーザーピンセットで使用するために作った料理で分離された網膜細胞のメッキと終わりで始まり。これらの料理は、細胞の接着剤の半分と細胞の撥半分に分かれています。細胞の接着面は、私たちの細胞が増殖する際に基板を提供するサル- 1抗体、の層でコーティングされています。他の接着剤基材は、他の細胞型を使用することができる。細胞の撥側は、ポリ- HEMAの薄層で被覆されている。皿のポリ- HEMA側に播種した細胞は、レーザーピンセットでトラップ運ばれ、ペアを作成するにはSAL - 1側のセルに隣接して配置されています。任意のサイズの細胞群の形成は、この手法で可能なはずです。 &quot;レーザーピンセット制御マイクロマニピュレーションは、&quot;研究者は細胞が移動するかを選択できることを意味し、そして細胞間の所望の距離は、標準化することができる。レーザービームは、培養皿の透明な表面を通過するので、セルの選択と配置は、同封の、無菌環境で行われます。細胞は、ビデオタイムラプスによって監視し、必要な細胞生物学的なテクニックを使用することができます。この手法は、細胞間相互作用の調査を助けるかもしれない。

Watch this Scientific Journal Video about Using Laser Tweezers For Manipulating Isolated Neurons In Vitro at JoVE.com

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

このビデオでは、in vitroでレーザーピンセットを用いた培養神経細胞の操作を説明します。

体外で分離された神経細胞を操作するためのレーザーピンセットを使用して

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult ...

using-laser-tweezers-for-manipulating-isolated-neurons-in-vitro

Research

JoVE Journal

Biology

10.3K ビュー.  University of Medicine and Dentistry of New Jersey - UMDNJ. このビデオでは、in vitroでレーザーピンセットを用いた培養神経細胞の操作を説明します。

ビデオ: Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

FMラベリングを用いた神経細胞の測定エキソサイトーシス

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

生物学

Silicon Microchips for Manipulating Cell-cell Interaction

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from development to malignant transformation.&nbsp; In particular, interaction with neighboring stroma has been implicated in a plethora of biological phenomena; however, conventional techniques limit the ability to interrogate the spatial and dynamic elements of such interactions.
In Micromechanical Reconfigurable Culture (RC), we employ a micromachined silicon substrate with moving parts to dynamically control cell-cell interactions through mechanical repositioning. Previously, this method has been applied to investigate intercellular communication in co-cultures of hepatocytes and non-parenchymal cells, demonstrating time-dependent interactions and a limited range for soluble signaling 1.
Here, we describe in detail the preparation and use of the RC system. We begin by demonstrating the handling of the device parts using tweezers, including actuating between the gap and contact configurations (cell populations separated by a narrow 80-&micro;m gap, or in direct intimate contact). Next, we detail the process of preparing the substrates for culture, and the multi-step cell seeding process required for obtaining confluent cell monolayers. Using live microscopy, we then illustrate real-time manipulation of cells between the different possible experimental configurations. Finally, we demonstrate the steps required in order to regenerate the device surface for reuse: toluene and piranha cleaning, polystyrene coating, and oxygen plasma treatment.

This article describes an experimental approach for dynamic regulation of cell-cell interactions between adherent cells on a micrometer scale. Manipulation of intercellular communication between hepatocytes and stromal cell is demonstrated. The developed platform enables investigation of cell-cell interactions in a variety of biological processes, including development and pathogenesis.

細胞間相互作用を操作するためのシリコンマイクロチップ

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from ...

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

診断ツールを開発するマイクロエレクト​​ロメカニカルシステム（MEMS）の使用

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

使用シュワン-アストロサイト相互作用の解析インビトロアッセイ

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

リボソーム結合した新生ポリペプチドの分離<em&gt; in vitroで</em&gt; mRNAに沿って並進一時サイトを識別するために

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

高リン酸化タウタンパク質を用いたインビトロ凝集アッセイにおいて

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

マウス涙腺を操作する

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

マウス単離された灌流腎臓テクニック

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

スペクトラルフローサイトメトリーにより固形組織から単離された細胞懸濁液の分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.