我々は、このプロトコルが最初に設計され、その研究室で、シカゴ大学で博士アーロンP.フォックスに感謝、そしてプロトコルのさらなる発展のためのウィリアムローデン（セントルイス大学）。 私たちは、ホワイトホール財団と支援のための国立科学財団に感謝します。

パート1：炭素繊維の調製<ol><li>キャピラリーピペットの〜1.25 ×長さは長さの炭素繊維（我々は7μm径を使用する）の束をカット。 </li><li> 〜コンテナ内のアセトン100mLの沸騰によるサイジングを外し、30分以上熱い、沸騰アセトンに繊維を追加。 </li><li>暖かいアセトンから繊維を取り出し、そして新鮮なアセトンの50〜100 mLできれいなビーカーに移す。 </li><li>オープン150ミリメートルペトリ皿とairdry一晩にバンドルを配置することにより乾燥した繊維。 </li></ol>パート2：火 - 研磨ガラス毛細管<ol><li>鉗子を中心にチューブを保持し、約flame.Rotateチューブに端を公開することにより、（我々は長いマイクロヘマトクリット毛細管は1.5mm外径、1.3ミリメートル内径、7.5センチメートルを使用）各キャピラリーチューブを火 - ポーランド炎の中に3回。 </li><li>チューブを逆さにして、再度3回回転させ、炎に反対側の端を置きます。 </li><li>ガラス皿にファイアーポリッシュチューブをセットし、彼らは炭素繊維で満たされることの準備が整うまで、オーブンに移す。 </li></ol>第3部：キャピラリーチューブにカーボン繊維をスレッディング<ol><li>マチャドらの説明に似て。 （2008年）とMundroffとワイトマン（2002）は、フィルタを真空に接続されているプラ​​スチック製のチューブを使用してガラス管に繊維を吸う。 </li><li>チューブの繊維の外のscalpel.Leave ¼インチを使用して繊維をカット。 </li></ol>パート4：電極プラーでピペットファイバを引っ張る<ol><li>電極プラー（我々はナリシゲ日本モデルPP - 830を使用）にキャピラリーチューブを置き、場所にキャピラリチューブを保持するためにノブを締めて。 </li><li>最適な引き手の設定で設定され、二人プル実行するためにスイッチを操作する。 </li><li>ピペットが半分に引っ張られると、ハサミを使って中心にベアファイバを切断して二つの部分を分ける。 </li><li>各電極の半分を削除し、繊維の1 / 8インチのチューブの外に残されるように、はさみでガラスseal.Trimの繊維を壊すために突き出したファイバーを引っ張る。 </li><li>約16繊維電極が引き出されるまで繰り返し、1〜4を繰り返します。 </li></ol>第5部：エポキシグラスのシールを作る<ol><li>ドラフトで6分間55℃に設定したホットプレート上でエポキシの既製のアリコートを加熱する。 </li><li>エポキシは、ラップを加熱している間〜束に引っ張ら電極16を​​テープを使用して。 </li><li>エポン樹脂が6分間加熱された後、ホットエポン樹脂（14％、w / v）の0.7グラムXOF MPDAを追加。バイアルを渦巻くことによりミックス。 </li><li>エポキシの十分な量が毛細管現象を介してガラスのヒントを入力する、20秒程度のホットエポキシプラス硬化剤のsolution.Afterに電極の束をつけます。 </li><li>スロットで段ボールにバンドル、場所の電極を元に戻すと、オーブンでこのトレイを置く、100℃に設定F. </li><li>少なくとも48時間後、、エポキシ樹脂で埋めるこわれた先端を持って、、それぞれの電極を検査段ボールホルダーの所定の位置に戻すとバック​​、複数のファイバー、etc.Afterを持っていなかった解剖microscope.Discard電極の下に各電極を調べるoven.Electrodesに通常製造後約1ヵ月に適しています。 </li></ol>第6部：炭素繊維を切断<ol><li>テフロンテープでスライドガラスの上に慎重に炭素繊維電極を置きます。 </li><li>解剖顕微鏡の下に先端を可視化する。 </li><li>細胞に対する炭素繊維を配置するための平坦な清浄な表面を残すために炭素繊維を垂直に横切っカットするクリーンな手術用メスの刃を使用してください。 </li></ol>

<ol>
	<li>Mundorf, M. L., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amperometry+and+cyclic+voltammetry+with+carbon+fiber+microelectrodes+at+single+cells.">Amperometry and cyclic voltammetry with carbon fiber microelectrodes at single cells.</a> Curr Protocols in Neurosci. 6 (14), 1-6 (2002).</li><li>Machado, D. J., Montesinos, M. S., Borges, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Good+practices+in+single-cell+amperometry.">Good practices in single-cell amperometry.</a> Methods in Molec. Biol. 440, 297-313 (2008).</li></ol>

このプロトコルは、電流滴電極を生成し、カットする方法について説明します。電流測定の録音中に、炭素繊維の電極は、分泌細胞の表面に配置されます。エキソサイトーシス活性は、カテコールアミンの酸化後の電子の移動によって生じる電気化学的電流を示す電流測定スパイクとして観測される。練習は作るとアンペロメトリック電極を切断すると、ノイズレベルを大幅に市販の電極の?...

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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>Epoxy Hardener 1,3-phenylene-diamene flakes</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>P23954-100G04 716AJ</td>
<td>Aliquots are stored in a dessicator and should not be exposed to light.</td>
</tr>
<tr>
<td>Carbon Fibers</td>
<td>Material</td>
<td>Goodfellow</td>
<td>C005725</td>
<td>&nbsp;</td>
<tr>
<td>Micro-Hematocrit Capillary Tubes</td>
<td>Material</td>
<td>Fisher</td>
<td>NC9836768</td>
<td>&nbsp;</td>
<tr>
<td>Epoxy Resin</td>
<td>Reagent</td>
<td>Miller-Stephenson</td>
<td>Epon Resin 828</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>Instrument</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Electrode Puller</td>
<td>Instrument</td>
<td>Narishige Japan</td>
<td>PP-830</td>
<td>1st pull = 24.8 degrees C; 2nd pull = 62.7 degrees C</td>
</tr>
<tr>
<td>Oven</td>
<td>Instrument</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>set to 100 degrees F.</td>
</tr>
<tr>
<td>Hot plate</td>
<td>Instrument</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>set to 55 degrees C</td>
</tr>		</tbody>
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fabrication of amperometric electrodes

炭素繊維電極は、電流測定の測定のための単一細胞内小胞からのカテコールアミンの放出を検出するために重要です。ここで、我々は、低ノイズ（&lt;0.5 Pa）の電極を生成するために必要なテクニックを説明します。技術は、以前の研究（1,2）によって発行された記述から変更されています。電極は、炭素繊維を作製し、繊維を吸引するフィルターで真空を使用して、各キャピラリーチューブに個別にスレッドによって行われます。次に、繊維と毛細管は微尖った先端で二つの部分、それぞれを作成し、電極プラーによってプルアップされています。電極はエポキシガラスのシールを作成するための硬化剤と混合して熱い、液状エポキシ樹脂に浸漬されています。最後に、電極はエポキシ樹脂を硬化するオーブン内に配置されます。電極を慎重に取り扱い、それらが一貫して損傷することなく行われることを保証することが重要です。このプロトコルは、単一細胞から記録するための電流測定電極を作製し、カットする方法を示しています。

Watch this Scientific Journal Video about Fabrication of Amperometric Electrodes at JoVE.com

Fabrication of Amperometric Electrodes

このプロトコルは、炭素繊維電極を生成する方法について説明します。電極は、その後、炭素繊維のアンペロメトリーと小胞からのカテコールアミン放出を検出するために使用されます。

アンペロメトリー電極の作製

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

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Analytical Chemistry

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Electrochemical Analyses and Redox Titration

SCIENTISTS IN ACTION

14.3K ビュー.  Saint Louis University School of Medicine. このプロトコルは、炭素繊維電極を生成する方法について説明します。電極は、その後、炭素繊維のアンペロメトリーと小胞からのカテコールアミン放出を検出するために使用されます。

ビデオ: Fabrication of Amperometric Electrodes

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

慢性実験における実装電極やマイクロダイアリシスプローブ用光重合型歯科用接着性レジンの応用

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

生物学

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues.  In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates.  The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive.  Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule.  Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks.    Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.  Micropatterning of several cell types was demonstrated on these substrates.   In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

光学的に透明な酸化インジウムスズ電極上にパターニングセル

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular ...

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

ニューロン相馬と軸索のコンパートメントのためのマイクロ流体デバイスの作製

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

PDMS Device Fabrication and Surface Modification

PDMSデバイスの作製と表面改質

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

マイクロ流体デバイスの低コスト作製のためのPDMSの非プラズマボンディング

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

Fabrication of the Thermoplastic Microfluidic Channels

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using polymer/nanoparticle composite microscale lysis and solid phase extraction columns. The recovered samples are concentrated, small volume samples that are PCRable, without any additional cleanup. Here, we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then, we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns. We demonstrate grafting and polymerization of a carbon nanotube/polymer composite column for bacterial cell lysis. We then show the lysis process followed by solid phase extraction of nucleic acids from the sample on chip using a silica/polymer composite column. The attached protocols contain detailed instructions on how to make both lysis and solid phase extraction columns.

Here we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns.

熱可塑性マイクロ流体チャネルの作製

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using ...

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades. Today, such pipettes are made from glass capillaries using a programmable puller. Such instruments heat the capillary using either a metal filament or a laser and draw out the glass using gravity, a motor or both. Pipettes for patch-clamp recording are formed using only heat and gravity, while sharp electrodes for intracellular recording use a combination of heat, gravity, and a motor. The procedure used to make intracellular recording pipettes is similar to that used to make injection needles for a variety of applications, including cRNA injection into Xenopus oocytes. In general, capillary glass &lt;1.2 mm in diameter is used to make pipettes for patch clamp recording, while narrower glass is used for intracellular recording (outer diameter = 1.0 mm). For each tool, the puller is programmed slightly differently. This video shows how to make both kinds of recording pipettes using pre-established puller programs.

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

パッチピペットおよびプログラマブルプラーとシャープ電極作成

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades.  Today, such pipettes are made from glass capillaries ...

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. Lightweight recording devices carrying a high density array of tetrodes allow for the simultaneous monitoring of the activity of tens to hundreds of individual neurons. Here we describe a protocol for the fabrication of a micro-drive array with twenty one independently movable micro-drives. This device has been used successfully to record from hippocampal and cortical neurons in our lab. We show how to prepare a custom designed, 3-D printed plastic base that will hold the micro-drives. We demonstrate how to construct the individual micro-drives and how to assemble the complete micro-drive array. Further preparation of the drive array for surgical implantation, such as the fabrication of tetrodes, loading of tetrodes into the drive array and gold-plating, is covered in a subsequent video article.

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

In this protocol we demonstrate how to fabricate a micro-drive array for chronic electrophysiological recordings in rats.

慢性のためのマイクロドライブアレイ in vivoで録音：ドラ​​イブの作製

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

筋原培養組織コンストラクトの作製

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

接着細胞培養用の酸素挿入の作製と動作

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...