マイクロ流体チャネルは、環状ポリオレフィン（ゼオネックス® 690R、ゼオンケミカル（株）、ルイビル、ケンタッキー州）で製造されています。ゼオネックスは、多孔質モノリスの形成に使用される光の指示化学に不可欠である、136 ° Cのガラス転移温度（T g）を持ち、透明なUVです。マイクロチャネルは、マイクロ流体プラットフォームの負の（逆）機能を持っているニコ電鋳マスターモールドとのマイクロホットエンボス加工によって製造される。ホットエンボス加工は、次のとおり行われます。 <ol><li>金型およびポリマー基板は、2つの平行な金属のプラテンの間に配置されています。 </li><li>プラテンは、ゼオネックスのT gを上記の30 º Cであるエンボス加工温度（166℃）、まで加熱されています。 </li><li>プラテンは、その後約5分間一緒に押され、圧力は250 psiで維持されます。 </li><li>モールドと基板は、その後1〜2分のために冷却し、加熱されたプラテンから削除し、手動で互いに分離される。 </li><li> 1.5 mm径の井戸は、サンプル導入および収集のためのチャネルの両端で掘削されています。 </li><li>エンボス加工プラスチック基板は、熱的に同封のマイクロ流体チャネルを形成するために、ゼオネックスの別の部分で接着されている。エンボス加工基板とカバー部分は、UV -オゾンやシール効率を向上させ、密閉チャネル構造（はなはだとKlapperich、2007）の再現性を保証するために、前に熱接合にプラズマ処理することができます。 </li></ol>プラスチック製マイクロ流体チャネル内の固相抽出（SPE）カラムの作製固相の作製は、2段階のプロセスです。最初は、製造されたマイクロチャネルは、プラスチック製のデバイスにSPEカラムの密着性を向上させるために表面修飾される。表面の光重合を経由してグラフト重合する高分子マイクロチャネルの壁を官能化するために使用されます。 移植の手順は以下の通りです： <ol><li>マイクロチャネルは、水素抽象化する光開始剤である3％のベンゾフェノン、とエチレンジアクリレート（EDA）とメチルメタクリレート（MMA）の1:1混合物で充填される。 EDA、MMA及びベンゾフェノンはSigma - Aldrich、セントルイス、MOから購入されています。 </li><li>チップは、紫外線露光器（CL - 1000 UV架橋剤、UPV社、アップランド、カリフォルニア州）で254 nmの紫外波長と200ミリジュール/ cm 2のエネルギーで10分間後、UV照射です。接ぎ木は、H -抽象化重合と第二段階におけるモノリスの調製に用いられる重合混合物内に取り込まなくては表面テザーポリマー鎖、の結果を介して行われます。 </li><li>過剰のモノマーを真空吸引を使用して、メタノールの15μLを洗浄することによりチャネルから削除されます。 </li></ol>グラフトプロセスの後、微孔質ポリマーモノリスは、DNAの抽出のための捕捉シリカ粒子そのマイクロチャネル内に形成される。 多孔質モノリスは、以下のように その場で調製されています。 <ol><li>表面改質されたチャンネルはBuMA（15重量％）、EDMA（10％重量）、1 - ドデカノール（52.5重量％）、シクロヘキサノール（22.5重量％）、DMPAPから成るモノリス前駆体の混合物に関して、（1重量％で充填される単量体）と0.7μmのシリカ粒子（プレポリマー混合物の合計量に対して15％）まで。 MO、セントルイス、シリカ微小球はPolysciences社（ウォリントン、PA）から購入し、モノマーとporogenic溶剤はSigma - Aldrichから購入されています。 </li><li>チップは、重合を開始、1.1分、200ミリジュール/ cm 2でUVクロスリンカーでUVを照射され、そして過剰のモノマーを真空吸引を使用して、メタノールの15μLを洗浄することによりチャネルから削除されます。 </li><li>流体接続（NanoPorts TM接続、アップチャーチ科学、オークハーバー、ワシントン州）は、その後、マイクロ流体チャネルの導入と収集井戸の上に貼付されています。 </li><li>多孔質モノリスは、100mL/hourの流量でシリンジポンプを使用して少なくとも30分間、メタノールで再び洗浄した。 </li></ol>プラスチック製マイクロ流体チャネル内のCNT溶解モノリスの作製 CNT（カーボンナノチューブ）溶解モノリスの作製は、2段階のプロセスです。最初に、作製したマイクロチャネルは、プラスチック製のデバイスでのSPEカラムの場合と同じ方法で表面改質（接木）です。 グラフトプロセスの後、微孔質ポリマーモノリスは、多層カーボンナノチューブを含浸されるマイクロチャネル内に形成される。 多孔質モノリスは、以下のように その場で調製されています。 <ol><li>多層ナノチューブは、2.27Mの濃度でシクロヘキサノールに再懸濁する。この再懸濁は、超音波処理細胞内分泌かく乱（超音波処理S - 250と振幅の50％と50％のデューティサイクルで30分間超音波処理され超音波ブランソン、ダンベリー、コネチカット州）製D、。超音波処理は、CNTの効果的かつ安定した0.5Mサスペンションを提供した。 CNTはNanolabs（ブライトン、MA）から購入した。 </li><li>表面改質されたチャネルは、シクロヘキサノールのその今のソリューション+ CNTが使用されているを除いて同様のモノリス前駆体の混合物で充填される。モノリスの混合物はBuMA（15重量％）、EDMA（10％重量）、1 - ドデカノール（52.5重量％）、およびシクロヘキサノール+カーボンナノチューブ（22.5重量％）で構成されています。この溶液に、光開始剤のDMPAPは（モノマーに対して重量1.13パーセント）が追加されます。モノマー、porogenic溶剤とDMPAPAはSigma - Aldrich、セントルイス、MOから購入されています。 </li><li>チップは、重合を開始するために0.9分、120ミリジュール/ cm 2でUVクロスリンカーでUVを照射する。デバイスはcrosslikerで180度反転、120 MJ / cm 2の別の0.9分間照射する。過剰のモノマーを真空吸引を使用してメタノール50μLで洗浄することによりチャネルから削除されます。 </li><li>流体接続（NanoPorts TM接続、アップチャーチ科学、オークハーバー、ワシントン州）は、その後、マイクロ流体チャネルの導入と収集井戸の上に貼付されています。 </li></ol> CNT溶解モノリスで使用するための細菌サンプルの調製細菌のサンプル用のサンプル調製には、チャネルの目詰まりを避けるために、サンプルが極端にそう集中されていないことを確認するために600nmの光学濃度の測定を行う伴います。 <ol><li>メディアの中の細菌と600nmの時のODの読み値を取得、これは暗順応計（エッペンドルフ暗順応計、エッペンドルフサイエンティフィック社、ウェストベリー、ニューヨーク州）で行われます。 ODの測定値が範囲0.18〜0.25 Aの内に収まるまで、サンプルを希釈する</li><li> 5分間6500 rpmでサンプルを遠心し、上清をデカントする。 </li></ol>溶解<ol><li> GuSCN * + 0.01パーセントSDS + 4％プロテイナーゼK（0.8mg/ml）で再懸濁します細菌。軽くボルテックスして（メディアの中の細菌1mlの1ミリリットルGuSCNに再懸濁しする必要があります）。プロテイナーゼKは、細菌のDNAに接続されているとDNAasesとRNAasesを抑制するタンパク質を変性を支援するために溶液に添加した。プロテイナーゼKの別の利点は、それはまた、グラム陰性細菌は、通常より多くのタンパク質を持っているものの、グラム陽性菌およびグラム陰性細菌の細胞壁の両方がタンパク質を大量に（含まれているため、望ましい細胞壁のタンパク質を分解することで助けることです。 ）。カオトロピックバッファは、さらに界面活性剤は細胞壁を破壊しながらタンパク質を変性とプロテイナーゼKを助けます。プロテイナーゼKとグアニジンチオシアネート（GuSCN）はキアゲン社（バレンシア、カリフォルニア州）から購入した。 SDSは、（ドデシル硫酸ナトリウム）ピアース（ロックフォード、IL）から購入した。 </li><li>すぐにPEEKチューブに接続してシリンジでサンプルをロードする。空気がシステムに存在しないようにシリンジを吸引除去する。 nanoportとチャネルにチューブを接続します。 </li></ol> KDS100シリンジポンプ（KDサイエンティフィック、ホリストン、MA社製）を実験に使用され、使用される流量は、すべてのステップを450μL/時間ですされました。 450ul/hrでのサンプル溶解のチャネルを流れる。 <ol><li>サンプルを収集し、SPEを経由してDNAの抽出（DNA抽出プロトコールを参照）に進みます。注意：懸濁液をGuSCNされているため、この細菌のサンプルは、負荷ステップ2を実行する必要はありません* </li></ol> DNA抽出プロトコル抽出操作は3ステップで構成されています： <ol><li>負荷。 </li><li>ワシントン州</li><li>溶出。 </li></ol> KDS100シリンジポンプ（KDサイエンティフィック、ホリストン、MA社製）を実験に使用され、使用される流量は、すべてのステップのための300μL/時間であった。 負荷<ol><li>条件の実験を開始する前に1〜2分間カオトロピックバッファ（GuSCN *、グアニジニウムチオシアネート）を使用してチャネル。 </li><li> 1:1の比率でカオトロピックバッファーでサンプルを混ぜる。 </li><li> 3分間チャネルを介してフローサンプルの混合物。 </li></ol>洗う<ol><li> 2分間70％エタノールを使用してチャネルを洗ってください。 </li><li> 2分間100％エタノールを使用してチャネルを洗ってください。 </li></ol>溶出する<ol><li> 3分間ミリポア水で抽出したDNAを溶出する。精製、PCR用DNAの15μLを収集する。 </li></ol> *キアゲンのキット（バッファRLT）からGuSCNが使用されました。

<ol>
	<li>Bhattacharyya, A., Klapperich, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+and+chemical+analysis+of+plasma+and+ultraviolet-ozone+surface+treatments+for+thermal+bonding+of+polymeric+microfluidic+devices.">Mechanical and chemical analysis of plasma and ultraviolet-ozone surface treatments for thermal bonding of polymeric microfluidic devices.</a> Lab Chip. 7, 876-882 (2007).</li></ol>

The authors have nothing to disclose.

fabrication of the thermoplastic microfluidic channels

私たちの研究室では、我々が正常に直接ポリマー/ナノ粒子複合材料のマイクロ溶解し、固相抽出カラムを用いて、ヒト血液、尿や便のマイクロリットルとsubmicroliterボリュームから核酸を単離した。回収したサンプルは、追加のクリーンアップを行わず、PCRableな集中、少量のサンプルです。ここでは、ホットエンボス加工とヒートシールを使用して熱可塑性マイクロ流体チップを作製する方法を示します。その後、我々は複合固相カラムを形成するために密封されたチップを介してその場の光監督接ぎ木面や重合で使用する方法を示します。私たちは、細菌の細胞溶解のために移植し、カーボンナノチューブ/ポリマー複合列の重合を実証。私たちは、その後、シリカ/ポリマー複合カラムを用いてチップ上のサンプルからの核酸の固相抽出に続いて溶解処理を示しています。添付のプロトコールは、溶解し、固相抽出カラムの両方を作成する方法に関する詳細な指示が含まれています。

Watch this Scientific Journal Video about Fabrication of the Thermoplastic Microfluidic Channels at JoVE.com

Fabrication of the Thermoplastic Microfluidic Channels

ここでは、ホットエンボス加工とヒートシールを使用して熱可塑性マイクロ流体チップを作製する方法を示します。その後、我々は、複合固相カラムを形成するために密封されたチップを介してその場の光監督接ぎ木面や重合で使用する方法を示します。

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 ...

fabrication-of-the-thermoplastic-microfluidic-channels

Research

JoVE Journal

Biology

13.3K ビュー.  Boston University. ここでは、ホットエンボス加工とヒートシールを使用して熱可塑性マイクロ流体チップを作製する方法を示します。その後、我々は、複合固相カラムを形成するために密封されたチップを介してその場の光監督接ぎ木面や重合で使用する方法を示します。

ビデオ: Fabrication of the Thermoplastic Microfluidic Channels

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 ...

生物学

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 ...

Microfluidic Applications for Disposable Diagnostics

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

使い捨て診断用マイクロ流体アプリケーション

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new ...

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

アフリカツメガエルの卵母細胞で発現イオンチャネルのパッチクランプ記録

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

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 ...

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 ...

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

P2X2リガンド依存性陽イオンチャネルと相互作用するタンパク質を特定するためのプロテオミクス

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

マイクロ流体プローブ：局在表面処理のための操作と使用方法

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

異種哺乳動物細胞で発現したイオンチャネルの変異導入と機能解析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

イオンチャネル研究のためのパッチクランプおよび灌流のテクニックで表さアフリカツメガエル卵母細胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...