この作品は、ドイツ学術振興協会（DFG）、フリッツ·ティッセン·財団とシャリテ/ MDCからの補助金によって賄われていた。

1。動物の麻酔と臓器摘出<ol><li>ケタミン（200 mg / kg体重）及びキシラジン（20 mg / kg体重）の腹腔内（ip）注射によりマウスをAnaesthetize。 </li><li> anticoagulateする血液凝固および血栓形成を避けるために250 IUのヘパリンipを注入します。 </li><li>深い昏睡状態に到達するまで反射消失を特徴とする、待つ。そっと尾つまんで角膜やテスト飛行反射をタッチすることで反射消失、角膜反射テストで確認するには。 </li><li>手術台の上にマウスを移し、仰臥位で固定してください。 </li><li>皮膚や剣状下腹壁を切開し、二つ折りの開胸を行います。肋骨弓に沿って両側にカットを伸ばし、その後内側腋窩線のリブをカットし、胸郭を上方に偏向させる。 </li><li> 、心を開き、大血管の位置を確認します。優しく良い大動脈を表示するために心臓の尾を押してください。ピンセットを用いて大動脈をクランプします。 </li><li> sのペアの凹部に心を置く<html> cissorsと1シングルカットと接続しているすべての血管を解剖する。ランゲンドルフカニュレーションのため上行大動脈の十分な大きさの部分を維持することを確認します。 </li><li>冷たい氷とプリoxygenized溶液1（ソリューションについては、 表1を参照）を充填したシャーレに直ちに摘出した心臓を転送します。 </li></ol> 2。ハートとランゲンドルフ灌流の調製<ol><li> 1.8F鋼カニューレと大動脈カニューレを挿入。空気塞栓を避けるようにしてください。 </li><li>溶液1の1ミリリットルと外科用縫合糸とフラッシュ冠状動脈と大動脈カニューレ上を固定します。 </li><li>ランゲンドルフ装置でカニューレを接続します。 </li><li>胸からランゲンドルフ挿管までの時間が心筋に長時間虚血/再灌流傷害を避けるために120秒を超えないようご注意ください。 </li><li> Ca 2 +の10mlの灌流心臓+ freeの溶液2（4ミリリットル/分）。 </li><li> 8分間（4 ml /分）のコラゲナーゼ溶液3を灌流心臓。 </li></ol>心腔のLE &quot;&gt; 3。顕微解離<ol><li>低Ca 2 +溶液4を含む予め温めておいた100mmのペトリ皿に心を移す。 </li><li>慎重にはさみで大動脈弁および他の非心臓組織を除去し、それを捨てる。 </li><li>別個の心房と心室とは別に、各チャンバに進みます。溶液4に浸細胞を維持。少量（未満5ミリリットル）を使用してください。 </li></ol> 4。心筋細胞のさらなる解離<ol><li>心房心筋： <ol><li>個別化する心房心筋細胞は別あらかじめ温めておいた100mmの培養皿に心房を転送し、優しく細かい鉗子でそれを引き離すを通して組織を解離させる。組織のほぼ完全解離を確認します。 </li><li> 5分間溶液5を1mlの細胞を中断するには拡大されたファイアーポリッシュプラスチックピペットチップで1mlのピペットを使用しています。 </li><li>セルフィルター（200μmのメッシュサイズ）を使用して、破片から細胞を分離。 </li> &lt;LI&gt; 5 mlの細胞懸濁液への解決策5、室温で16×gで2分間遠心分離器を追加します。 </li><li>次の手順は、細胞培養、ボンネットの下に運営されています。上清を捨て、溶液6 5mlにペレットを再懸濁する。 </li><li> 15 mlのチューブに10分間、重力によって沈降した後、室温で16×gで1分間遠心します。上清を取り除きます。溶液6の1-5 mlにその量に応じて細胞を再懸濁する。 </li></ol></li><li>心室心筋細胞： <ol><li>溶液4の5ミリリットルの微細鉗子との利害の左心室領域を解剖。 </li><li>ほとんどの細胞が分離されるまで、ゆっくりピペッティングにより細胞を一時停止します。 50 mlチューブに（200μmのメッシュサイズ）フィルタリング後に細胞溶液を移し、25ミリリットルの容量を追加します。 </li><li>室温で16×gで2分間遠心分離します。 </li><li>次の手順は、細胞培養、ボンネットの下に運営されています。上清を除去し、25 mlにペレットを再懸濁溶液6と10分間細胞の沈降が可能になります。 </li><li>細胞をカウントし、上清を除去し、25を追加 - 50ミリリットルの溶液6を細胞に。 </li></ol></li></ol> 5。細胞電気生理学、生化学的またはIF研究のための心筋細胞の調製<ol><li>電気生理学的研究のために、50 mlチューブに溶液6の心筋細胞を維持し、沈降を抑制する。 </li><li>ラミニンコート細胞培養ディッシュ（最終濃度PBS中に20μg/ mlのラミニン）の生化学的研究、 例えばカルシウムイメージング、板筋のために。 </li><li>免疫蛍光染色用ガラスカバースリップとラミニン溶液（最終濃度PBS中で50μg/ mlのラミニン）でコーティングを有する細胞培養皿プレートを準備します。 <ol><li>メッキ筋前に溶液を除去します。顕微鏡を用いて、プレート細胞と対照細胞密度。 </li><li> 2％CO 2中37℃で1時間のスリップ℃をカバーするために筋細胞が付着してみよう、溶液を除去および標準染色手順プロトコルで直ちに開始します。 </li><li>室温で10分間、PBS中の固定剤、 例えば、4％PFA（pH7.5）を用いてインキュベートし、5分間ごとに3 PBSでの洗浄工程に従ってください。 </li><li>細胞を透過すると非特異的結合抗体が10％血清、0.3％トリトン、室温で30分間、PBS中の0.2％BSAを用いて心筋細胞をインキュベート阻害する。 </li><li> 37℃で1時間のために一次抗体とインキュベート℃、前述したように洗ってください。 </li><li>室温で1時間、二次抗体とインキュベートします。核を対比染色し、α-アクチニン使用DAPI蛍光色素と共役ファロイジン（のAlexa Fluor 488、Invitrogen）に。 </li><li>洗浄後、ガラスカバーが蛍光封入剤でシラン処理された顕微鏡スライドとembed細胞上に慎重に滑る移す。 </li></ol></li></ol> 6。細胞の電気生理学<ol><li>周波数にホールセルパッチクランプ記録を行うshly室温で心房と心室の心筋細胞を単離した。 </li><li>外浴溶液の定義されたボリュームでいっぱい灌流チャンバーに健康現れる心筋細胞を移す。 117 mMのNaCl、4mMののKCl、1mMのKH 2 PO 4、4mMのNaHCO 3を 、1.7 mMのMgCl 2、3mMののCoCl 2、10mMのHEPES、10mMグルコース、0.02 mMのテトロドトキシン（TTX）、液（pH7.4） 。 pH値調整のためNaOHを使用してください。 </li><li>適切なパッチクランプ装置（ すなわち IX71倒立顕微鏡、Multiclamp 700Bアンプ、pClamp10.3ソフトウェア（Molecular Devices）を持つDigidata 1440A収集システムとPC）を使用します。 </li><li> 130のKCl、2mMのMgCl 2、11mMのHEPES、11mMのEGTA、5mMののNa 2のATP、0.4mMの硫酸ナトリウム 、GTP、5mMののNa 2 CPを含む細胞内液で満たされたときMΩ3-5の抵抗とパッチピペットを使用して、および4.9 mMのCaCl 2（pH7.2）で。 pH値調整のためにKOHを使用します。 </li><li>外向きK +電流デュを呼び起こす20ミリ秒、-40 mVにプレパルス後に70mVの保持電位から10 mV刻みで-60から+50 mVの電位をテストするためのリングは4.5秒の電圧ステップ。 </li><li>リーク電流は常に&lt;100 pA以下であることを確認します。 </li><li> 、Heteropodatoxin 2（HpTx2; Alomone）やテトラエチルアンモニウム（TEA;シグマ）;異なるK +電流は、4 - アミノピリジン（ICNバイオメディカル、アーバイン、カリフォルニア州、米国の4-AP）などの特異的阻害剤を使用するの解離ストック溶液は、細胞外浴溶液で調製し、 &quot;ベースライン&quot;のレコーディング後にマイクロチップを介して細胞のできるだけ近い付近に直接適用されるべきである。平衡化は &quot;薬物&quot;のレコーディングの前に2〜3分間認められるべきである。 </li><li>分析のためのセルサイズに個々の細胞（全細胞膜容量）の電流振幅を正規化します。 pClamp10.3ソフトウェア（Molecular Devices社）または同等のソフトウェアを使用して、オフラインのデータを分析します。 </li></ol>

<ol>
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我々は、開示することは何もない。

遺伝子欠失に関連する心機能と心臓の病理を研究する遺伝子改変マウス系統の成長発展に伴い、in vitroで特定の遺伝子欠失の影響を研究するための特殊なメソッドへの関心の高まりもある。我々の研究室では、心臓の再分極にKvをチャネルの補助サブユニットのファミリーの役割を研究しています。 KCNE遺伝子はヒト心室と心房の再分極11日、15日に重要な役割を果たして?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td></tr><tr><td>テトロドトキシン</td><td> Alomone </td></tr><tr><td> 4 - アミノピリジン</td><td> ICNバイオメディカル</td></tr><tr><td> Heteropodatoxin 2 </td><td> Alomone </td></tr><tr><td>テトラエチルアンモニウム</td><td>シグマケミカル</td></tr><tr><td>コラゲナーゼタイプ2 </td><td>ワージントン</td></tr></tbody></table>

isolation and kv channel recordings in murine atrial and ventricular cardiomyocytes

KCNE遺伝子は、それらの機能的特性を変更するためにKvをチャネルαサブユニットとヘテロ複合体を形成したKvチャネルの補助サブユニットの小さな家族のためにエンコードします。 KCNE遺伝子の変異は、QT延長症候群および/または心房細動などの不整脈を有する患者で発見されている。しかし、これらの疾患につながる正確な分子病態は不明なままである。以前の研究では、これらの遺伝子に変異を引き起こす疾患の電気生理学的特性は、主に異種発現系において研究されてきたと報告された影響が直接ネイティブ心筋細胞に変換することができれば、我々は確認することはできません。私たちの研究室では、したがって、異なるアプローチを使用しています。我々は可視化実験誌のこの号で記述する独特の技法-我々は、直接細胞電気生理学によってノックアウトマウスから単離した心筋細胞におけるKCNE遺伝子欠失の影響を研究する。 geneticaから心LLY KCNE組み換えマウスが急速に大動脈カニュレーションにより摘出し、ランゲンドルフ装置に搭載されている。心筋内の遊離Ca 2 +が EGTAによってバインドされており、心筋細胞の解離は、次に特化した低Ca 2 +を含むバッファーコラゲナーゼと冠状動脈の逆行灌流することによって達成される。心房、右心室自由壁と左心室は、その後、顕微技術によって分離することができる。カルシウムは、その後徐々に洗浄操作を構成する複数のステップで孤立した心筋細胞に戻されます。ノー自発収縮との健全な外観の心房と心室の心筋細胞は、その後直ちに分離後の最初の6時間以内にパッチクランプ法や他の生化学的解析により電気生理学的解析に供される。

in vitroでの関心のある特定の遺伝子の機能を研究するために遺伝子組み換えマウスから成体マウスの心筋細胞の単離は、さらに心臓の病態生理を理解するための強力なツールとなっています。このメソッドは現在、世界中で基本的な科学研究室の唯一の小さいながらも増加によって使用されています。しかし、成体マウス心室の心筋細胞の単離は、トリッキーなことが、経験豊富な手?...

Watch this Scientific Journal Video about Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes at JoVE.com

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KVチャネル機能不全は、心臓不整脈に関連付けられています。我々はKvをチャネルの補助サブユニットのノックアウトマウスからの心房と心室の心筋細胞を単離するための体系的なプロトコルを利用するような不整脈を引き起こす分子メカニズムを研究するために。隔離された心筋細胞は、その後すぐに携帯電気生理学的研究、生化学的または免疫蛍光法（IF）のアッセイのために使用することができます。

マウス心房と心室の心筋細胞における分離とKvチャンネル録音

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

isolation-kv-channel-recordings-murine-atrial-ventricular

Research

JoVE Journal

Biology

13.3K ビュー.  Charité Medical Faculty and Max-Delbrück Center for Molecular Medicine (MDC). KVチャネル機能不全は、心臓不整脈に関連付けられています。我々はKvをチャネルの補助サブユニットのノックアウトマウスからの心房と心室の心筋細胞を単離するための体系的なプロトコルを利用するような不整脈を引き起こす分子メカニズムを研究するために。隔離された心筋細胞は、その後すぐに携帯電気生理学的研究、生化学的または免疫蛍光法?...

ビデオ: Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

マウス造血幹細胞およびリネージュコミット前駆細胞の表現型の解析と分離

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

生物学

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

新生仔マウス心筋細胞の単離および培養

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

構造と機能研究のための脂質膜へのKvチャネルの再構築

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

マウス心筋細胞の単離と生理解析

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

細胞シグナリングのための成体マウスの心筋細胞の単離と文化<em&gt; in vitroで</em&gt;心肥大

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

一チャネルセル付パッチクランプ法

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

精製された野生型および変異型CFTRタンパク質の機能的再構成とチャネル活性の測定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

培養HL-1心房筋細胞単層の電圧とカルシウムデュアルチャネル光学マッピング

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

成体マウスの心筋細胞の単離、培養および形質導入

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a homogenous population of cells, the iPSC-CMs generated by current differentiation protocols represent a mixture of cells with ventricular-, atrial-, and nodal-like phenotypes, which complicates phenotypic analyses. Here, a method to optically record action potentials specifically from ventricular-like iPSC-CMs is presented. This is achieved by lentiviral transduction with a construct in which a genetically-encoded voltage indicator is under the control of a ventricular-specific promoter element. When iPSC-CMs are transduced with this construct, the voltage sensor is expressed exclusively in ventricular-like cells, enabling subtype-specific optical membrane potential recordings using time-lapse fluorescence microscopy.

Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein.