加えて、我々は彼らの優れた技術サポートのために人間の心房組織とクラウディアLiebetrau、カトリンKupserとラモナナーゲルの提供のためにハイデルベルク大学で心臓外科医に感謝します。のCa 2 +トランジェント測定の確立中に彼の役立つ提案やアドバイスのためにもアンディ·W·トラフォードに感謝します。彼らは、細胞電気生理学と心筋細胞分離における基本的なテクニックやスキルを学ぶために私たちを提供する機会をドレスデン工科大学：著者は、薬理学および毒物学の部門（ウルスラレイブンズ頭）のメンバーに彼らの最も深い感謝の意を表したい。 著者の研究は、ドイツ研究財団（Do769/1-1-3）、心房細動コンピテンスネットワークを通じて教育研究のドイツ連邦環境省（01Gi0204）と循環器病研究のためのドイツ語センターを通じて欧州連合によってサポートされていますEuの心房細動におけるトランスレーショナルリサーチのためropeanネットワーク（EUTRAF、FP7-HEALTH-2010、大規模な統合プロジェクト、提案番号261057）および欧州·北米の心房細動·リサーチ·アライアンス（ENAFRA）財団Leducq（07CVD03）の付与。 ビデオファイルに示す概略図はセルヴィエ医療技術を用いて製造された。

実験プロトコルは、ローカル倫理委員会によって承認される必要があり、すべての患者は書面によるインフォームドコンセントを与える必要があります。我々の研究は、ハイデルベルク大学（＃2011-216N-MA）、医学部マンハイムの倫理委員会によって承認され、人類の福祉のためにすべての、制度的、国内および国際的なガイドラインに準拠して行った。すべての患者は書面によるインフォームドコンセントを与えた。 0。ヒト心房組織の取得心肺バイパス移植のための心臓切開手術を受ける患者におけるルーチンカニュレーション手順の間、右心耳の先端部は、通常、除去され、心房心筋細胞の単離に使用することができる。筋細胞Cを防止、BDM、収縮抑制剤、切除後の組織サンプルは、2,3 -ブタンジオンモノオキシムを（ 表I含む滅菌のCa 2 +を含まない輸送溶液を50ミリリットルファルコンチューブにすぐに転送されますontracture）。 30と45分の間の輸送時間で、我々は孤立した心房心筋細胞の数と質の両方に対する冷却または酸素化の明確な利点を認識しませんでした。研究室への一般的な輸送にはできるだけ迅速にする必要があります。長い輸送時間が避けられない場合は、酸素溶液中で4℃輸送℃で有利かもしれない。 1。 Prearrangements <ol><li>ジャケット付ビーカー（ 表II）の温度を制御thermocirculator、スイッチオン。ビーカー内の温度が37℃に等しいことを確認してください全体の分離プロセス全体ビーカー内の温度を一定に維持するために、ガラス蓋付きビーカーをカバーしています。 </li><li> 3ガラス製ビーカーにコラゲナーゼIおよびプロテアーゼXXIVを秤量し、室温で保管してください。酵素は、使用直前に希釈することができる。 </li><li>のCa 2 20mlのを保存+ 4でペトリ皿に自由溶液℃の</li><li>のCa 2ストア250ミリリットル37℃の水浴中で+自由溶液℃に</li></ol> 2。ティッシュの清掃<ol><li>ペトリ皿（〜直径10cm）に輸送ソリューションと一緒に組織サンプルを転送し、大体ハサミを使用して脂肪組織を削除します。 </li><li>組織試料を秤量する。 200〜600 mgの細胞単離のために使用される。残りの組織が保存生化学分析のために液体窒素中で凍結することができる。 </li><li> 20ミリリットルのCa 2 + 4で無料のソリューション°Cを含むペトリ皿に組織サンプルを転送し（ステップ1.3を参照）、約1mm 3サイズでの小さなチャンクに組織サンプルをチョップ。 </li><li>次の手順は、100％O 2、37℃で、連続処刑の下に実行されるべきである。ピペットチップは、強力な泡立ちを避けるために、連続した空気流を生成するために、酸素チューブに配置することができる。 </li><li>中のCa 2 +を含まない溶液と一緒にティッシュの塊を転送ジャケットビーカーへと磁気攪拌棒で約3分間注意深くかき混ぜる。 </li><li>攪拌を停止し、組織の塊は数秒のために落ち着くことができます。ナイロンメッシュ（200ミクロン、 表II）を通して上清を慎重にひずみ。メッシュから鉗子を使用してビーカーに拘束され、組織の塊を返します。 </li><li>リフィルのCa 2ビーカー+送料液及び洗浄工程を2.4と2.5を2回繰り返す。 </li></ol> 3。最初の酵素消化<ol><li>コラゲナーゼやプロテアーゼを含んだ20ミリリットル酵素溶液E1（ 表I）で組織の塊を再停止し、10分間注意深くかき混ぜる。 </li><li> 20μMのCa 2 +の最終濃度を得るために、10のCaCl 2溶液を40μLを加える。 </li><li> 35分後には、組織の塊のほとんどがビーカーに残るような方法でナイロンメッシュ（200ミクロン、 表II）を注意深く上清を負担。残りを返すメッシュからビーカーに組織塊を雨が降った。 </li></ol> 4。第二酵素消化<ol><li>私はわずか20ミリリットル酵素溶液E2含むコラゲナーゼ（ 表I）で再び組織の塊を再懸濁します。 20μMのCa 2 +の最終濃度を得るために、すぐに10のCaCl 2溶液を40μLを加える。 </li><li>第二消化中さらに時折組織血栓をチョップするはさみを使用しています。 </li><li> 5分後、細胞の解離をチェックするパスツールピペットを使用してサンプルを取る。棒状、横紋心筋が表示されるまで、この2〜3分ごとに繰り返します。 </li><li>攪拌を停止し、組織の塊が約20〜30秒間、落ち着くことができます。 </li><li> 50ミリリットルファルコンチューブ（チューブ）にナイロンメッシュ（200ミクロン、 表II）を注意深く上清を負担。メッシュからビーカーに拘束され、組織の塊を返します。 </li><li> 20ミリリットルストラでビーカー内の組織チャンクを再懸濁さらにGE溶液（ 表I）10とディスペンサーと20ミリリットル血清ピペットを用いて穏やかな機械的粉砕により細胞を解離する。気泡が細胞の生存と品質に有害であるので、気泡の形成を避けるようにしてください。 </li><li> 50ミリリットルファルコンチューブ（チューブB）にナイロンメッシュ（200ミクロン、 表II）を注意深く上清を負担。 </li></ol> 5。決勝のCa 2 +濃度の最終準備と調整<ol><li> 10分間、95×gでファルコンチューブAとB（ステップ4.5および4.7を参照）の両方を遠心します。 </li><li>吸引により慎重に両方のチューブから上清を取り除きます。ペレットを乱さないようにしてください。上清を捨てる。 </li><li> 1.5 mlのストレージソリューション（ 表I）各（室温）の両方でペレットを再懸濁する。 </li><li> 2回ずつファルコン〜10のCaCl 2溶液の7.5μLを加え、慎重にかき混ぜる。各ステップの後に10分間インキュベートする。 </li><li>0.2mMの最終のCa 2 +濃度を得るために10のCaCl 2溶液15μlのを追加します。 </li></ol> 6。蛍光灯のCa 2 +指示薬フルオ-3 AM（ 図1）と筋細胞のロード<ol><li> 2 mlのマイクロ遠心チューブ（エッペンドルフチュー​​ブ）内に細胞懸濁液1.5mlの（チューブおよび/またはチューブB）に移す。 </li><li>次の手順は、蛍光のCa 2 +インジケーターフルオ-3の光感度を考慮して実行する必要があります。 </li><li>プルロニックF-127（ 表III）原 ​​液（20％無水DMSO中w / v）で取得するための44μlのフルオ-3の膜透過性のアセトキシメチルエステル誘導体（フルオ-3 AM、 表III）50μgのを溶かす1mMのフルオ3、最大で1週間、-20℃で保存することができる原液、AM。 </li><li> 15μlのを追加フルオ-3は1.5の細胞懸濁液（ステップ6.1を参照）を振とうを含むマイクロチューブに原液AM慎重に。 </li><li>光学的に不透明なボックスで10分間細胞懸濁液をインキュベートする。 </li><li>簡単に言えば、約6,000 rpmで遠心。 </li><li>上清を捨て、1.5 mlの浴溶液（ 表IV）でペレットを再懸濁する。 </li><li>実験を開始する前に脱エステル化のために約30分間、細胞懸濁液にしておきます。 </li></ol> 7。同時パッチクランプと落射蛍光Ca 2 +の測定パッチクランプ測定は、この見直しの主要な話題ではないので、我々はこの技術の深さの説明に多くを提供する他の出版物への興味を持った読者を参照してください。11-14完全を期すために、我々は測定するためのプロトコルの簡単な要約を提供する活動電位またはL型Ca 2 +電流は、同時のCa 2 +過渡録音と一緒に両方。 実験中筋細胞を風呂solutiと37℃で灌流さ急速灌流システム（Octaflow IITM、ALA科学機器、NY）を用いて、（ 表IV）で。電圧クランプ実験のために、K +電流を浴溶液に4 -アミノピリジン（5ミリモル/ L）及び塩化バリウム（0.1ミリモル/ L）を添加することによってブロックされる。ホウケイ酸ガラス微小電極が使用され、ピペット溶液（ 表V）で満たされたときMΩ2-5の抵抗を傾けるしている必要があります。フルオ-3に加えて（ステップ6を参照）筋細胞のローディングAM、フルオ3はピペット溶液（ 表V）に含まれている。蛍光は488 nmで励起して放出された光は、（&lt;520 nm）での[Ca 2 +] iは仮定に変換されます<img alt="式（1）" fo:content-width="1.540in" fo:src="/files/ftp_upload/50235/50235eq1highres.jpg" src="/files/ftp_upload/50235/50235eq1.jpg" /> ここで、k Dフルオ-3（864のn =解離定数M）、F =フルオ-3蛍光;各実験の終了時に得られたF = 最大のCa 2 + -飽和蛍光12の両方の電気信号及び落射蛍光のCa 2 +信号が同時に記録される。活動電位は1.2倍しきい値強度の1ミリ秒電流パルスを使用して、現在のクランプモードで0.5 Hzで刺激される。 L型Ca 2 +電流は、100ミリ秒に続いて、高速のNa +電流を不活性化するために-40 mVまで-80 mVの、100ミリ秒ランプパルスの保持電位を用いた電圧クランプモードで測定されている0.5 Hzで+10 mVのにテストパルス。

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J. 80 (01), 1915-1925 (2001).</li><li>Worthington, K., Worthington, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Worthington Enzyme Manual. , (1993).</li><li>Christ, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology-specific+effects+of+the+IKur/Ito/IK,ACh+blocker+AVE0118+on+ion+channels+in+human+chronic+atrial+fibrillation.">Pathology-specific effects of the IKur/Ito/IK,ACh blocker AVE0118 on ion channels in human chronic atrial fibrillation.</a> Br. J. Pharmacol. 154, 1619-1630 (2008).</li><li>Dobrev, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+downregulation+of+G-protein-coupled+inward+rectifying+K++current+IK,ACh+in+chronic+human+atrial+fibrillation:+decrease+in+GIRK4+mRNA+correlates+with+reduced+IK,ACh+and+muscarinic+receptor-mediated+shortening+of+action+potentials.">Molecular basis of downregulation of G-protein-coupled inward rectifying K+ current IK,ACh in chronic human atrial fibrillation: decrease in GIRK4 mRNA correlates with reduced IK,ACh and muscarinic receptor-mediated shortening of action potentials.</a> Circulation. 104, 2551-2557 (2001).</li><li>Hatem, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+compartments+of+sarcoplasmic+reticulum+participate+in+the+excitation-contraction+coupling+process+in+human+atrial+myocytes.">Different compartments of sarcoplasmic reticulum participate in the excitation-contraction coupling process in human atrial myocytes.</a> Circ. Res. 80, 345-353 (1997).</li><li>Heidbuchel, H., Vereecke, J., Carmeliet, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+different+potassium+channels+in+human+atrium.+Contribution+to+the+basal+potassium+conductance.">Three different potassium channels in human atrium. Contribution to the basal potassium conductance.</a> Circ. Res. 66, 1277-1286 (1990).</li><li>Hove-Madsen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrial+fibrillation+is+associated+with+increased+spontaneous+calcium+release+from+the+sarcoplasmic+reticulum+in+human+atrial+myocytes.">Atrial fibrillation is associated with increased spontaneous calcium release from the sarcoplasmic reticulum in human atrial myocytes.</a> Circulation. 110, 1358-1363 (2004).</li><li>Molina, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+adenosine+monophosphate+phosphodiesterase+type+4+protects+against+atrial+arrhythmias.">Cyclic adenosine monophosphate phosphodiesterase type 4 protects against atrial arrhythmias.</a> J. Am. Coll. Cardiol. 59, 2182-2190 (2012).</li></ol>

ワージントン生化学社は、ビデオファイルの生産コストをカバーすることによって、この出版物を支持した。資金提供者は、研究デザイン、データ収集と分析、公表することを決定、または原稿の準備で何の役割を持っていた。

ここでは、心臓切開手術を受けた患者から得られた右心房付属器からのヒト心房筋細胞の単離のための方法が記載されている。細胞質のCa 2の測定のために、これらの心筋細胞を使用するために、我々は+貯蔵溶液からEGTAを省略して、以前に記載された方法4-11に適合。 すでに1970年には筋細胞が+消化中のCa 2の存在下で解離しているが、それらのすべては、したがって拘縮および非生存。16,17にあったことが観察された、細胞単離のCa 2 +を含まない溶液中で行われる。しかし、のCa 2の生理的濃度の再導入は急速+ Ca 2 +の流入および細胞死をもたらした。これは、もともとはジマーマンとフルスマンによって灌流心の中で観察されたCa 2 +のパラドックス現象として記載されている。7.0のpHの低下、など、分離培地の18修正&lt;22のCa 2 +パラドックスを防止するために提案されているストレージを含む溶液EGTAで&gt; 19タウリン20又はCa 2 +少量の添加（ステップ3.2および4.1を参照のこと）、21ならびに単離された筋細胞の記憶（商標）17は、しかし、のCa 2 +が EGTAを介してバッファリングのCa 2 +過渡二相崩壊におけるL型Ca 2 +電流誘起のCa 2 +過渡振幅および結果の振幅を減少させることは周知である。23そこで、EGTAを省略典型的なプロパティと相性減衰でのCa 2 +トランジェントを得るために全体の分離プロセス全体。のCa 2から細胞を保護するために+パラドックス我々は、0.2 mMのまで段階的にストレージソリューションの最終のCa 2 +濃度を増加させた。 コラゲナーゼの選択は、おそらく成功した筋細胞分離のための最も重要なステップです。従来のコルagenasesはクロストリジウムヒストリチカムから得られた粗製の製剤であり、他のプロテイナーゼ、polysaccharidasesとリパーゼの数に加えて、コラゲナーゼを含んでいます。彼らの一般組成のコラゲナーゼに基づいて、異なるタイプに細分化されます24ワージントンコラゲナーゼ·タイプIとIIが正常に我々の現在記述されたプロトコルでは、人間の心房筋細胞を単離する。4-10,15,25-30ために使用されてきた私たちは、コラゲナーゼの使用をお勧めします我々はまた、コラゲナーゼタイプIIを使用して生細胞の許容量を得ることができたものの、Iを入力します。しかしながら、単一のコラゲナーゼタイプ内の酵素活動に関する重要なバッチ間変動がある。これらのバリエーションは、慎重なバッチの選択と分離手順を最適化するための様々なバッチのテストが必要。ワージントン生化学株式会社（からオンラインで利用可能なバッチ·選択ツール<a href="http://www.worthington-biochem.com/cls/match.php" target="_blank">http://www.worthington-bio</a>chem.com / CLS / match.php）は、ヒト心房筋細胞を単離するのに適していることが示された組成物で使用可能なバッチを見つけるために使用することができる。現在、我々は、コラゲナーゼは、250 U / mgのコラゲナーゼ活性を345 U / mgでcaseinase活動、2.16 U / mgのクロストリパイン活性および0.48 U / mgのトリプシン活性（ロット＃49H11338）Iを入力してください。 本稿に記載された手順を用いて得られた細胞をパッチクランプ研究のCa 2 +トランジェント測定およびまた両方図15の組合せに対して8時間以内に使用することができ、これらの細胞は電場刺激に応答して細胞の収縮の測定を可能パッチクランプピペットを（未発表の観察）を使用して、または電気刺激。

パッチクランプ法と細胞のCa 2探査による心臓イオンチャネルの電気生理学的特性を研究+異常が孤立した心筋細胞を必要とする処理。これらは通常、消化酵素に対する心臓組織サンプルのインビトロ曝露（コラゲナーゼ、ヒアルロニダーゼ、ペプチダーゼ等）後に得られる。 1955 2で実行可能な心筋細胞の単離の最初の報告以降のプロトコルの多量は、マウス、ラット、ウサギ、イヌ、モルモットおよびヒトを含む様々な種から単心房および心室の心筋細胞を収穫するために開発されている。このレビューでは、人間の心房筋細胞の分離に焦点を当てています。我々はワージントン生化学社、USA（が提供する&quot;ワージントン組織解離ガイド&quot;を参照して他の種からの心筋細胞を単離するための手順について<a href="http://www.tissuedissociation.com" target="_blank">www.tissuedissociation.com</a> ）。 らによって記載されている方法から派生しています。3ここでは、するために以前に公表されたメソッドから構成されている技術は、のステップバイステップの説明を提供パッチクランプ実験のためだけでなく、同時に細胞内Ca 2 +測定のためだけでなく、適切な心房筋細胞を得る。4-11

<table border="1">
 <tbody>
 <tr>
 <td> &nbsp;</td>
 <td> &nbsp;</td>
 <td> &nbsp;</td>
 <td>Transport solution</td>
 </tr>
 <tr>
 <td>Albumin</td>
 <td>Sigma-Aldrich</td>
 <td>A3059</td>
 <td>Storage solution: 1%</td>
 </tr>
 <tr>
 <td>BDM</td>
 <td>Sigma-Aldrich</td>
 <td>31550</td>
 <td>Transport solution: 30</td>
 </tr>
 <tr>
 <td>DL-b-Hydroxy-butyric acid</td>
 <td>Sigma-Aldrich</td>
 <td>H6501</td>
 <td>Storage solution: 10</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>Sigma-Aldrich</td>
 <td>G8270</td>
 <td>Transport solution: 20 
 Ca2+-free solution: 20 
 Enzyme solution E1 and E2: 20 
 Storage solution: 10</td>
 </tr>
 <tr>
 <td>L-Glutamic acid</td>
 <td>Sigma-Aldrich</td>
 <td>G1251</td>
 <td>Storage solution: 70</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Merck</td>
 <td>1049360250</td>
 <td>Transport solution: 10 
 Ca2+-free solution: 10 
 Enzyme solution E1 and E2: 10 
 Storage solution: 20</td>
 </tr>
 <tr>
 <td>KH2PO4</td>
 <td>Sigma-Aldrich</td>
 <td>P5655</td>
 <td>Transport solution: 1.2 
 Ca2+-free solution: 1.2 
 Enzyme solution E1 and E2: 1.2 
 Storage solution: 10</td>
 </tr>
 <tr>
 <td>MgSO4</td>
 <td>Sigma-Aldrich</td>
 <td>M9397</td>
 <td>Transport solution: 5 
 Ca2+-free solution: 5 
 Enzyme solution E1 and E2: 5</td>
 </tr>
 <tr>
 <td>MOPS</td>
 <td>Sigma-Aldrich</td>
 <td>M1254</td>
 <td>Transport solution: 5 
 Ca2+-free solution: 5 
 Enzyme solution E1 and E2: 5</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Sigma-Aldrich</td>
 <td>S3014</td>
 <td>Transport solution: 100 
 Ca2+-free solution: 100 
 Enzyme solution E1 and E2: 100</td>
 </tr>
 <tr>
 <td>Taurin</td>
 <td>Sigma-Aldrich</td>
 <td>86330</td>
 <td>Transport solution: 50 
 Ca2+-free solution: 50 
 Enzyme solution E1 and E2: 50 
 Storage solution: 10</td>
 </tr>
 <tr>
 <td>Collagenase I</td>
 <td>Worthington</td>
 <td>4196</td>
 <td>Enzyme solution E1 and E2: 286 U/ml</td>
 </tr>
 <tr>
 <td>Protease XXIV</td>
 <td>Sigma-Aldrich</td>
 <td>P8038</td>
 <td>Enzyme solution E1 and E2: 5 U/ml*</td>
 </tr>
 <tr>
 <td>pH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Transport solution: 7.00 
 Ca2+-free solution: 7.00 
 Enzyme solution E1 and E2: 7.00 
 Storage solution: 7.40</td>
 </tr>
 <tr>
 <td>adjusted with</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Transport solution: 1 M NaOH 
 Ca2+-free solution: 1 M NaOH 
 Enzyme solution E1 and E2: 1 M NaOH 
 Storage solution: 1 M KOH</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Concentrations in mM unless otherwise stated. BDM, 2,3-Butanedione monoxime. *Protease XXIV is included in Enzyme solution E1 only.</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table I. Solutions.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nylon mesh (200 &mu;m)</td>
 <td>VWR-Germany</td>
 <td>510-9527</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Jacketed reaction beaker</td>
 <td>VWR</td>
 <td>KT317000-0050</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table II. Specific equipment.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dimethyl-sulphoxide</td>
 <td>Sigma-Aldrich</td>
 <td>D2650</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fluo-3 AM (special packaging)</td>
 <td>Invitrogen</td>
 <td>F-1242</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pluronic F-127</td>
 <td>Invitrogen</td>
 <td>P6867</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table III. Substances for loading of myocytes with Fluo-3 AM.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Bath solution</td>
 </tr>
 <tr>
 <td>4-aminopyridine*</td>
 <td>Sigma-Aldrich</td>
 <td>A78403</td>
 <td>Bath solution: 5</td>
 </tr>
 <tr>
 <td>BaCl2*</td>
 <td>Sigma-Aldrich</td>
 <td>342920</td>
 <td>Bath solution: 0.1</td>
 </tr>
 <tr>
 <td>CaCl2 &times; 2H2O</td>
 <td>Sigma-Aldrich</td>
 <td>C5080</td>
 <td>Bath solution: 2</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>Sigma-Aldrich</td>
 <td>G8270</td>
 <td>Bath solution: 10</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H9136</td>
 <td>Bath solution: 10</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Merck</td>
 <td>1049360250</td>
 <td>Bath solution: 4</td>
 </tr>
 <tr>
 <td>MgCl &times; 6H2O</td>
 <td>Sigma-Aldrich</td>
 <td>M0250</td>
 <td>Bath solution: 1</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Sigma-Aldrich</td>
 <td>S3014</td>
 <td>Bath solution: 140</td>
 </tr>
 <tr>
 <td>Probenecid</td>
 <td>Sigma-Aldrich</td>
 <td>P8761</td>
 <td>Bath solution: 2</td>
 </tr>
 <tr>
 <td>pH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Bath solution: 7.35</td>
 </tr>
 <tr>
 <td>adjusted with</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Bath solution: 1 M HCl</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table IV. Bath solution for patch-clamp. *4-aminopyridine and BaCl were included for voltage-clamp experiments only.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Pipette solution</td>
 </tr>
 <tr>
 <td>DL-aspartat K+-salt</td>
 <td>Sigma-Aldrich</td>
 <td>A2025</td>
 <td>Pipette solution: 92</td>
 </tr>
 <tr>
 <td>EGTA</td>
 <td>Sigma-Aldrich</td>
 <td>E4378</td>
 <td>Pipette solution: 0.02</td>
 </tr>
 <tr>
 <td>GTP-Tris</td>
 <td>Sigma-Aldrich</td>
 <td>G9002</td>
 <td>Pipette solution: 0.1</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H9136</td>
 <td>Pipette solution: 10</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Merck</td>
 <td>1049360250</td>
 <td>Pipette solution: 48</td>
 </tr>
 <tr>
 <td>MgATP</td>
 <td>Sigma-Aldrich</td>
 <td>A9187</td>
 <td>Pipette solution: 1</td>
 </tr>
 <tr>
 <td>Na2ATP</td>
 <td>Sigma-Aldrich</td>
 <td>A2383</td>
 <td>Pipette solution: 4</td>
 </tr>
 <tr>
 <td>Fluo-3**</td>
 <td>Invitrogen</td>
 <td>F3715</td>
 <td>Pipette solution: 0.1</td>
 </tr>
 <tr>
 <td>pH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>7.20</td>
 </tr>
 <tr>
 <td>adjusted with</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>1 M KOH</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table V. Pipette solution for patch-clamp*. *On experimental days pipette solution is stored on ice until use. **Fluo-3 is added from a 1 mM stock solution on experimental days.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation of human atrial myocytes for simultaneous measurements of ca2+ transients and membrane currents

パッチクランプ法と心臓細胞のCa 2 +の処理異常の探査と心臓イオンチャネルの電気生理学的特性の研究では、孤立した心筋細胞を必要とします。さらに、これらの技術を用いて患者から心筋細胞を調査する可能性は、例えば心房細動（AF）などの心臓疾患の分子基盤を解明する貴重な要件である。1は、ここでは両方に適しているヒト心房筋細胞の単離のための方法を記載パッチクランプ研究と細胞内Ca 2 +濃度の同時測定。まず、開胸手術を受けた患者から得られた右心耳は、小さな組織の塊（ &quot;チャンク法&quot;）に切断し、+を含まない溶液のCa 2で洗浄されています。その後、組織の塊を20μMのCa 2 +とソリューションを含むコラゲナーゼおよびプロテアーゼで消化されています。その後、隔離された筋細胞はハーヴです組織懸濁液の濾過と遠心分離によりSTED。最後に、セル記憶溶液中のCa 2 +濃度を0.2mMの段階的に調整される。我々は簡単に+とCa 2 +の分離の過程でバッファリングし、また活動電位と膜電流の代表的な録音を提供し、両方一緒に同時のCa 2 +トランジェント測定、これらの孤立した筋細胞で行わでのCa 2の意味を議論する。

図2Aは、単離されたヒト右心房筋細胞から3つの代表的な例を示しています。我々はCellFinder顕微鏡スライド（上の細胞懸濁液（ステップ5.5）10μlのピペットで細胞収率定量化する<a href="http://www.antenna.nl/microlab/index-uk.html" target="_blank">http://www.antenna.nl/microlab/index-uk.htmlを</a> ）。 16.5±3.1 cells/10液（nは= 29）対5.1±2.3 cells/10μL： 図2Bにおいて平均細胞の収量は明らかに慢性AF（CAF）が低いセルの利回りの傾向患者サンプル（チューブがあることを示すそれぞれSRとCAFで（N = 10）、P &lt;0.05;チューブB：17.9±3.9 cells/10液（N = 29）対5.9±SRとCAFで2.0 cells/10液（N = 9）、それぞれに、p = 0.107）。 活動電位の測定および細胞質のCa 2 +過渡電流の同時録画の代表例を図3に示されている。調べた細胞、Tの約90％で彼はアクション電位トリガのCa 2 +放出が明確と通常細胞の収縮を引き起こす。既報の通り、細胞の整合性のために認められた指標である静止膜電位は、-73.9平均約±2.7 mVの（N = 10分の23筋/患者）及び-77.7±1.8 mVの（N = 8分の19筋/患者）SRとCAFであった（P&gt; 0.05）。15 図4は、電位依存性L型Ca 2 +電流と細胞質のCa 2 +トランジェントの代表、同時録画を示しています。非選択的β-アドレナリン受容体アゴニストイソプレナリン（1μM）の適用は、無傷のβ-アドレナリン作動性シグナル伝達カスケードを示唆し、私はCa、Lと細胞質のCa両方の振幅2 +トランジェントを向上させます。 <img alt="図1" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig1highres.jpg" src="/files/ftp_upload/50235/50235fig1.jpg" /> Figure 1。筋のFluo-3のフローチャートは、（ステップ6.1から6.5を参照）プロトコルをロードしています。 M / V、質量/体積。 <img alt="図2" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig2highres.jpg" src="/files/ftp_upload/50235/50235fig2.jpg" /> 図2。ストレージソリューションの1時間後、単離されたヒト右心房筋細胞。Bは、ストレージソリューションのセル10μlに数え細胞収率の±SEM（ステップ5.5を参照）を意味します。 nは、各グループ内製剤の数を指す。 *はp &lt;0.05。 <img alt="図3" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig3highres.jpg" src="/files/ftp_upload/50235/50235fig3.jpg" /> アクション電位トリガのCa 2洞調律から心房筋細胞における+トランジェント（CAT）とCHRの図3。代表レコーディングプロピオン心房細動の患者。トップ：刺激（0.5ヘルツ）に使用注入電流膜（I M）。下：膜電位（V M）、およびトリガ猫（下）の同時記録。 （フォークトらの許可を得て再プロット。2012）15 <img alt="図4" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig4highres.jpg" src="/files/ftp_upload/50235/50235fig4.jpg" /> L型Ca 2上イソプレナリン（1μM）の効果図4代表的録音+現在のトリガ型のCa 2洞調律、慢性心房細動の患者からの心房筋細胞における+トランジェント（CAT）。トップ：電圧クランププロトコル（0.5 Hz）です。下：現在の総純膜の同時記録（I M）、主にL型Ca 2 +電流（中央）とトリガ猫（下）を反映している。 （フォークトから許可を得て再プロット、ら2012年）15 <img src="/files/ftp_upload/50235/50235table1.jpg" alt="表1" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235table1highres.jpg" /> 表Iソリューション。 <img src="/files/ftp_upload/50235/50235table2.jpg" alt="表2" fo:content-width="2in" fo:src="/files/ftp_upload/50235/50235table2highres.jpg"/> 表II。特定の機器。 <img src="/files/ftp_upload/50235/50235table3.jpg" alt="表3" fo:content-width="2in" fo:src="/files/ftp_upload/50235/50235table3highres.jpg"/> 表III。フルオ-3と筋細胞のローディングのための物質がAM。 <img src="/files/ftp_upload/50235/50235table4.jpg" alt="表4" fo:conteNT-幅= &quot;2.5インチ&quot;のfo：SRC = &quot;/ files/ftp_upload/50235/50235table4highres.jpg&quot; /&gt; 表IV。パッチクランプ用浴溶液。 <img src="/files/ftp_upload/50235/50235table5.jpg" alt="表5" fo:content-width="2.5in" fo:src="/files/ftp_upload/50235/50235table5highres.jpg"/> パッチクランプ*表V.ピペット溶液。

Watch this Scientific Journal Video about Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents at JoVE.com

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

我々は、細胞内Caために使用することができ、ヒト心房筋細胞の単離を記載<sup&gt; 2 +</sup電気生理学的パッチクランプ研究との組み合わせで&gt;測定。

カルシウムの同時測定のための人間の心房筋細胞の分離<sup&gt; 2 +</sup&gt;過渡と膜電流

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

isolation-human-atrial-myocytes-for-simultaneous-measurements-ca2

Research

JoVE Journal

Biology

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

20.2K ビュー.  University of Duisburg-Essen. 我々は、細胞内Caために使用することができ、ヒト心房筋細胞の単離を記載 2 +測定。

ビデオ: Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

成体ラットの心筋細胞の初代培養

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

生物学

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

共焦点イメージングのための成人心筋細胞の単離と遺伝子操作

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

イメージングカルシウムのためのテクニック 2 +人間の精子でシグナリング

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

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 of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

ハイスループットマイクロプレート呼吸測定のためのマウスの骨格筋の最小量からミトコンドリアの単離

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

の開発と特性評価<em&gt;インビトロ</em&gt;微小血管ネットワークおよび内皮の定量的測定の[Ca<sup&gt; 2+</sup&gt;]<sub&gt;私</sub&gt;と一酸化窒素の生産

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

成体マウスから洞房結節筋細胞の単離、培養、および機能解析のための方法

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

蛍光顕微鏡による生細胞内のミトコンドリアのカルシウムとミトコンドリア膜電位の同時測定

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5&#180;-OH ends or 5&#180;-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5&#180;-end processing, DNA damage, and DNA repair.

Here we describe a method amenable to simultaneously quantitate and genome-wide map ribonucleotides in highly intact DNA at single-nucleotide resolution, combining enzymatic cleavage of genomic DNA with its alkaline hydrolysis and subsequent 5&#180;-end sequencing.

同時マッピングとヒトのミトコンドリア DNA のリボヌクレオチドの定量

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

細胞内カルシウムと新鮮な分離とそのままマウス脳血管内皮細胞の膜電位の同時計測

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...