この研究は教育科学技術省（2013068067）から資金提供を受け、韓国の国立研究財団（NRF）を介して基礎科学研究開発プログラムによってサポートされていました。脳韓国21大学院教育の韓国省の計画、科学技術、ソウル国立大学病院、高血圧の韓国学会（2013）、SKテレコム研究基金（なし。3420130290）と中国の国家自然科学基金からのことで（NSFC 31460265; NSFC 81260035）。

プロトコルは健康の国連国立研究所によって公開実験動物の管理と使用に関する指針に従っている（NIH公開番号85-23、1996年改訂）。これは、ソウル大学（IACUC承認番号：SNU-101213から1）の施設内動物管理使用委員会（IACUC）によって承認されました。 1.バッファーの調製（表材料および装置） <ol><li> NaCl、135;のKCl、5.4;のMgCl 2、3.5;グルコース、5; HEPES、5;のNa 2 HPO 4、0.4;タウリン、20; pH値：MMにおける実験（当日に新鮮な分離溶液300mlを準備7.4 NaOHで）。 </li><li> CaCl 2 0.2; MgSO 4を5;のKCl 5.4、NaClを120ナトリウム-ピルビン酸5; 5.5グルコース、タウリン20; HEPES 10;マンニトール29; pHは7.4：MMにおける実験（当日に新鮮なストレージソリューションの100ミリリットルを準備）NaOHで。 </li><li> ;のKCl、4;のNaH 2 PO 4、0.33;上記MgCのNaCl、141.4：mMの中の灌流液の1Lを（準備L 2は、1。 HEPES、10;グルコース、5.5; CaCl 2、1.8;マンニトール、14.5; NaOHでpHを7.4）。 </li><li>分離を25 mlのコラゲナーゼ（1 mg / mlで）、プロテアーゼ（0.1 mg / mlで）、ウシ血清アルブミン（BSA、1.67mg / mlで）、およびCa 2+（0.05ミリモル）を添加することによって、コラゲナーゼ溶液1を30mlの調製溶液。 </li><li>分離溶液16.7 mlに、BSA（1.67 mg / mlで）、およびCa 2+（0.05ミリモル）を、コラゲナーゼ（1mg / ml）を添加することによって、コラゲナーゼ溶液2を20ミリリットルを準備します。 </li><li>分離溶液40mlにBSAを0.4gを添加することにより、BSA溶液40mlを調製します。 2ビーカーに10ミリリットルと30ミリリットルを区切ります。 BSA溶液中の最終Ca 2+濃度を1mMになるようにBSA溶液を30mlまでのCa 2+を加えます。 </li></ol>左心室（LV）筋細胞の単離2.準備<ol><li>調製および単離の部屋に動物施設からクリーンな輸送ケージに8-12週齢、雄のSprague-Dawley（SD）ラットを転送します。 </li><li> H37℃。に2水浴を食べます 注：水のいずれかを使用水浴 - ジャケット付きリザーバとランゲンドルフ灌流システムの灌流チューブを。単一の筋細胞を分離するために、心筋組織のトランクを攪拌する他の水浴を使用してください。 </li><li>それぞれ、30ミリリットルと20ミリリットルにボリュームを補うために5ミリリットル及びコラゲナーゼ溶液1と16.7ミリリットルコラゲナーゼ溶液2の25ミリリットルに（のCa 2+なし）BSA溶液の3.3ミリリットルを追加します。 </li><li>それぞれ、ランゲンドルフ灌流システムにおける分離溶液（100ml）およびコラゲナーゼ溶液1（30mL）でカラム1（コロ1）および列2（コロ2）を追加する（ 図1A）。 </li><li>ランゲンドルフ灌流システム（ 図1A）中の酸素接続管を介して、大佐1とCol.2で分離ソリューションおよびコラゲナーゼ溶液1に酸素。同様に、含酸素コラゲナーゼ溶液2とを経由して 100％のO 2との振盪水浴中のBSA溶液酸素接続チューブ。 </li></ol> LV筋細胞の3分離<ol><li>ペントバルビタールナトリウム（30mg / kg）の腹腔内注射を介して、SDラットを麻酔し、ピンチつま先と撤退反射の欠如によって麻酔状態を確認。 </li><li>解剖トレイにラットを移動します。仰臥位では、テープで本体の側面に4脚を固定します。 </li><li>心を傷つけないように確実に、胸を開くために手術用ハサミで胸部半ば軸切開を適用します。接続血管（ 例えば、スーペリア、下大静脈、肺血管、および大動脈）と心膜膜13から心臓を解剖するためにきれいなハサミの別のペアを使用してください。 </li><li>細かい鉗子で大動脈をクランプ、 - （8ミリメートル5）、および急速に1分（ 図1A）内ランゲンドルフ灌流システムのカニューレを取り付けるのに十分な長さの大動脈の一部を残します。大動脈の上にしっかりと縫合糸4/0を接続します。 </li><li&gt;は大佐1のバルブの電源をオンにし、10分間予熱したと酸素分離液（灌流速度：12〜14ミリリットル/分）で分離された心臓を灌流。 、大佐1のバルブをオフにし大佐2のバルブをオンにして、8のためのコラゲナーゼ溶液1で心臓を潅流 - 10分。 </li><li>大動脈を切断することにより、消化心のマウントを解除し、新鮮な分離溶液（ 図1Bi）を含むフラスコに鉗子で大動脈を保持することによって、それを転送します。小さ ​​な断片（〜22ミリメートル、 図1Bii）に（中隔を含む）、LV自由壁の大部分をカットする細かいハサミやピンセットを使用してください。 </li><li>予熱したと酸素新鮮なコラゲナーゼ溶液2（ 図1Biii）を含むフラスコにピースを転送します。 10分間振ります。筋細胞を含むコラゲナーゼ溶液2に酸素を提供してください。 </li><li>吸引電球でドロッパーを用いて10 mlの遠心チューブにサスペンションおよびCa 2+を追加- -筋細胞を移動（BSA溶液を含みます1：ボリューム内の1）。 2分間の30グラムで遠心分離し、上清を捨てます。 BSA溶液5ml中の筋細胞ペレットを再懸濁します。上清を遠心分離し、廃棄します。 </li><li>筋細胞ペレットを分散し、RT（ 図1Biv-VI）で予備酸素ストレージソリューションの10ミリリットルで筋細胞を維持します。 </li><li>フラスコ内の残りのLV組織と - （3.9 3.7ステップ）の手順を繰り返します。 LV組織のほとんどは筋細胞の良好な収率を得るために消えるまで再び - （3.9 3.7ステップ）の手順を繰り返してください：注意してください。 </li><li>実験の終了まで室温で6-8時間のためのストレージソリューションに筋細胞を保管してください。 </li></ol>細胞内Ca 2+過渡状態と筋細胞の収縮の4同時測定<ol><li>アセトキシメチルエステルのFura-2 AM（2μM）でロードLV筋細胞。 注：ダークチューブ（ 図1Bvii）にロードされた筋細胞を持つすべてのロード手順と実験を行います。 <ol><li> CENTR10秒間2000×gで筋細胞懸濁液（1ml）にifuge。上清を捨て、低Ca 2+濃度（250μM、表材料および装置）とのタイロード溶液の1ミリリットル中の筋細胞ペレットを再懸濁。 </li><li>軽く筋細胞懸濁液を分散させる、のFura-2AMとポロキサマー407（2μl）を追加し、室温で混合物を維持- 15分間（20〜24 Oの C）（ 図1Bvii）。 </li><li>遠心分離は、混合物を5秒間、上清を廃棄し、500μMのCa 2+を含む灌流液の1ミリリットルで筋細胞ペレットを分散させます。 10分後、5秒間混合物を遠心分離し、上清を捨てます。 </li><li>新鮮な灌流液（500μMのCa 2+、1ミリリットル）を追加し、フラ- 2AM保つ-レコーディングのために、この溶液中にロードされた筋細胞ペレットを。 </li></ol></li><li> LV筋細胞の収縮と細胞内Caの測定2+ <ol><li>記録する前に、通る灌流チューブを埋めます36 O℃に予熱したあるタイロード溶液、水ジャケット</li><li> 8分 - 5のための倒立蛍光顕微鏡のチャンバーにロードされたLV筋細胞サスペンション - フラ2 AMの数滴を置きます。ゆっくりタイロード溶液（2.2ミリリットル/分）を灌流。 </li><li>フィールド刺激（2 Hz）を開始するデジタル刺激装置の前面パネルにある「スタート」ボタンを押してください。 注：出力電圧（10 V、5ミリ秒の持続時間）は、チャンバの両側に位置する白金線を介してチャンバ内の筋細胞に適用されます。 <ol><li>その記録のための契約を安定（ハイパーまたはハイポ収縮が表示されていないもの）筋細胞を選択します。 </li></ol></li><li>ビデオベースの筋節検出システムの水平位置に選択肢の筋細胞を調整し、サルコメアの最適な画像を得るために顕微鏡の焦点を調整します。明確な筋節が明らかに平均するまで観察されている領域に紫の長方形のボックスを配置しますサルコメア長（赤ピーク）の特異鋭いピーク（ 図2A、下の画像）が表示されます。 </li><li>フィールド刺激（ 図2AおよびB）に応答して、筋節長の変化を記録します。 注：1にロード筋細胞を使用してください - 2時間。 </li><li>ビデオベースの筋節検出システムのフィールドが筋細胞（ 図2A）の大きさになるようにカメラの絞りを調整します。 510 nmの（買収周波数千ヘルツ）で360ナノメートル/ 380 nmでの励起および発光で筋細胞を刺激します。録音サルコメア短縮とフィールド刺激（ 図2B）とFURA2 AM比。 </li></ol></li></ol>筋フィラメントのCa 2+感受性の5評価<ol><li>サルコメアの長さと定常状態での Ca 2+過渡電流の平均（10から20までのトレース）と（両方の測定値と同じ筋細胞の筋節長対のFura-2比の位相面のループをプロットsおよびデルタ変化; 図2C）。 </li><li>各プロットでは、50％の弛緩（EC 50、 図2C）でフラ-2比を定義します。各介入のループおよびEC 50の両方を比較してください。 </li></ol>

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The authors declare that they have no competing financial interests.

ここでは、単一の孤立心筋細胞における筋フィラメントのCa 2+感受性の変化を評価し、電気生理学的特性、細胞内Ca 2+過渡電流、および筋フィラメント動態と一緒に、このパラメータを測定することの重要性を強調するためのプロトコルを記述します。パラメータの一つまたは二つの記録は、心臓の収縮と弛緩のメカニズムを解明しない場合があるからです。個別に1?...

心臓の興奮収縮（EC）カップリングは、心筋の機械的特性を分析するための基本的なスキーム、 すなわち、心臓1,2の収縮機能です。 ECカップリングは、筋細胞膜のイオンチャネル（ 例えば、パッチクランプ技術によって測定することができる電位依存性Na +チャネル）の活性に対する二次膜の脱分極によって開始されます。 LTCCs を経由して 、その後の電位依存性L型の活性化のCa 2+チャネル（LTCCs）およびCa 2+流入は、ナノモル（nMでから細胞質ゾルCa 2+濃度を増加させ、リアノジン受容体（RyRs）を介してのCa 2+放出の大部分をトリガ）（μM）レベルをマイクロモル。細胞質ゾルの Ca 2+におけるこのような増加は、細いフィラメント中のトロポニンC（TnCの）へのCa 2+結合を促進し、誘発フィラメント複合体の立体構造変化をアクチン-ミオシン相互作用を容易にし、myocardiを達成しますアルは3を収縮しました。逆に、細胞質ゾルCa 2+が SR中のCa 2+ -ATPaseを介して筋小胞体（SR）（SERCA2aの）に戻し再取り込まれる又はNa + / の Ca 2+交換器と原形質膜を介して筋細胞の外に押し出され、 Ca 2+ ATPアーゼ1,2。その結果、細胞質ゾルのCaの減少は2+アクチン-ミオシンと筋細胞弛緩1-3の解離の結果、元の状態に戻し細いフィラメントの立体構造変化を扇動します。ほとんどの哺乳動物の心臓細胞における1細胞質ゾルのCa 2+の除去の90％ -それは70を占めるので、この方式では、SERCA2aの活性は、一般に、心筋弛緩の速度を決定すると考えられます。このように、などLTCC、のRyRとのSERCA2a、による異常のCa 2+ハンドリングが疾患のある心臓1-4で損なわれた収縮と弛緩のための主要なメカニズムと考えられてきました。 、2+総細胞内Caの約1％のECカップリング勘定のメッセンジャーおよびCa 2+の大部分として機能する細胞内Ca 2+バッファ5,6にバインドされている無料のサイトゾルの Ca 2+。これは、様々なのCa 2+緩衝液は、 例えば、膜リン脂質、ATP、クレアチンリン酸、カルモジュリン、パルブアルブミン、筋原繊維のTnC、ミオシン、SERCA2aの、かつSRのカルセケストリン心筋細胞に豊富であるという事実にある。5,6,7。これらの中でも、SERCA2aのとTnCのは、支配的なのCa 2+バッファ5,6,7です。また、Caは2+、そのバッファへの結合は単収縮時の動的なプロセスであり、追加の原因を結合のCa 2+の変化（ 例えば、Ca 2+を 30〜50％が2+トランジェント7のTnCに結 ​​合し、カルシウムの間にそれから解離します）濃細胞内Ca 2+の変更で、細胞質ゾルに遊離Ca 2+の結果を「解放」entration。その結果、細胞内Ca 2+レベルの摂動は、収縮不全および不整脈8,9の前駆体である異常な筋フィラメントの動きを誘導します。 （生理学的および病理学の両方）は、多くの要因が筋フィラメントのCa 2+バッファリングと筋フィラメントのCa 2+感受性8-10に影響を与える筋フィラメントタンパク質の転写後修飾の源とすることができます。最近では、のCa 2+トランジェント、異常のCa 2+放出、および不整脈8のポーズ依存増強を誘発、筋フィラメントタンパク質における突然変異は、Ca 2+結合親和性および細胞内Ca 2+ハンドリングを向上させることが報告されました。この考え方に沿って、我々はまた、神経型一酸化窒素合成酵素のアップレギュレーションに対する二次性高血圧ラット心臓における筋フィラメントのCa 2+の脱感作が上昇、拡張期と収縮期のCa 2+レベルに関連付けられていることが示されています今度は、のCa 2+依存性不活性化12 LTCCの脆弱性を増加させる11、。したがって、筋フィラメントのCa 2+感受性は、細胞内Ca 2+ホメオスタシスと筋細胞収縮機能の「アクティブ」な調節因子です。これは、筋フィラメントおよびCa 2+の間の相互作用、筋細胞、ECカップリング及び心臓機能の徹底的な調査のためのタンパク質の取り扱いを分析することが必要となってきています。 ここでは、孤立した心筋細胞における筋フィラメントのCa 2+感受性の変化を評価するプロトコルを記述します。細胞内Ca 2+プロファイル、筋フィラメントのCa 2+感受性および収縮の包括的な分析は、新規メカニズムを根底にある心筋の力学を発掘します。

<table border="0" cellpadding="0" cellspacing="0" width="1136">
	<tbody>
		<tr height="66">
			<td height="66" style="height:66px;width:315px;">Sprague Dawley rat</td>
			<td style="width:131px;">Koatech</td>
			<td style="width:125px;"></td>
			<td style="width:565px;">8-12 weeks</td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:315px;">Pentobarbital Sodium</td>
			<td style="width:131px;">Hanlim Pharmaceutical (Korea)</td>
			<td style="width:125px;">AHN901</td>
			<td style="width:565px;">Insurance code:645301220</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">NaCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S9625</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">KCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P4504</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">NaH2PO4</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S8282</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">HEPES</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">H3375</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Glucose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">G8270</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">MgCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">M2001</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Mannitol</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M4125</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:315px;">MgSO4</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M5921</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Sodium Pyruvate</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P2256</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Taurine</td>
			<td style="width:131px;">Merck</td>
			<td style="width:125px;">8.08616.1000</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Na2HPO4&#160;</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">71649</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Bovine Fetal Albumin</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">A7906</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Collagenase Type 2</td>
			<td style="width:131px;">Worthington</td>
			<td style="width:125px;">LS004177</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Protease</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P6911</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Fura-2 (AM)</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:125px;">F1221</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Pluronic F127 20% solution in DMSO</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:125px;">P3000MP</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Shaking Water Bath</td>
			<td style="width:131px;">Chang Shin Scientific</td>
			<td style="width:125px;">Model: C-108</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:315px;">IonWizard Softwae Suite</td>
			<td style="width:131px;">IonOptix Ltd</td>
			<td style="width:125px;">Experimental Builder</td>
			<td style="width:565px;">Acquisition and Analysis of EC Coupling Data in Myocytes</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:315px;">Myocyte Calcium &#38; Contractility Recording System</td>
			<td style="width:131px;">IonOptix Ltd</td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Circulating Water Bath</td>
			<td style="width:131px;">BS-Tech</td>
			<td style="width:125px;">BW2-8</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:315px;">Myocyte Fluorescence Microscope</td>
			<td style="width:131px;">Nikon</td>
			<td style="width:125px;">DIATPHOTO 200</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:315px;">MyoCam-S Power</td>
			<td style="width:131px;">IonOptix</td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="36">
			<td height="119" rowspan="3" style="height:119px;width:315px;">Fluorescence &#38; Video Detection</td>
			<td rowspan="3" style="width:131px;">IonOptix</td>
			<td style="width:125px;">MyoCam-S</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:125px;">CFA300</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:125px;">PMT400</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:315px;">Fluorescence &#38; System Interface</td>
			<td style="width:131px;">IonOptix</td>
			<td style="width:125px;">FSI700</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:315px;">Excitation Light Source</td>
			<td style="width:131px;">IonOptix</td>
			<td style="width:125px;">mSTEP</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:315px;">High intensity ARC Lamp Power supply</td>
			<td style="width:131px;">Cairn Reseach</td>
			<td></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:315px;">Filter wheel controller</td>
			<td style="width:131px;">IonOptix</td>
			<td style="width:125px;">GB/MUS200</td>
			<td></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;">Digital Stimulator</td>
			<td style="width:131px;">Medical Systems Corportion</td>
			<td>S-98 Mutimode</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Compositions of Experimental Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Name</td>
			<td style="width:131px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td style="width:565px;">Comments</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:315px;">Isolation Solution (pH: 7.4, NaOH)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">NaCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S9625</td>
			<td style="width:565px;">Concentration (mmol) 135</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">KCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P4504</td>
			<td style="width:565px;">Concentration (mmol) 5.4</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">HEPES</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">H3375</td>
			<td style="width:565px;">Concentration (mmol) 5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Glucose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">G8270</td>
			<td style="width:565px;">Concentration (mmol) 5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">MgCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">M2001</td>
			<td style="width:565px;">Concentration (mmol) 3.5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Taurine</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">CB2742654</td>
			<td style="width:565px;">Concentration (mmol) 20</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Na2HPO4&#160;</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">71649</td>
			<td style="width:565px;">Concentration (mmol) 0.4</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Storage Solution (pH: 7.4, NaOH)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">NaCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S9625</td>
			<td style="width:565px;">Concentration (mmol) 120</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">KCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P4504</td>
			<td style="width:565px;">Concentration (mmol) 5.4</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">HEPES</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">H3375</td>
			<td style="width:565px;">Concentration (mmol) 10</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Glucose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">G8270</td>
			<td style="width:565px;">Concentration (mmol) 5.5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;">Concentration (mmol) 0.2</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Mannitol</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M4125</td>
			<td style="width:565px;">Concentration (mmol) 29</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">MgSO4</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M5921</td>
			<td style="width:565px;">Concentration (mmol) 5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Sodium Pyruvate</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P2256</td>
			<td style="width:565px;">Concentration (mmol) 5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Taurine</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">CB2742654</td>
			<td style="width:565px;">Concentration (mmol) 20</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:315px;">Perfusion Solution (Tyrode solution, pH: 7.4, NaOH)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">NaCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S9625</td>
			<td style="width:565px;">Concentration (mmol) 141.4</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">KCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P4504</td>
			<td style="width:565px;">Concentration (mmol) 4</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">NaH2PO4</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S8282</td>
			<td style="width:565px;">Concentration (mmol) 0.33</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">HEPES</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">H3375</td>
			<td style="width:565px;">Concentration (mmol) 10</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Glucose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">G8270</td>
			<td style="width:565px;">Concentration (mmol) 5.5</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td>Concentration (mmol) 1.8&#160;&#160;&#160;&#160;&#160; For Fura 2AM loading, CaCl2 concentrations are 0.25 mM and 0.5 mM</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">MgCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">M2001</td>
			<td style="width:565px;">Concentration (mmol) 1</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Mannitol</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M4125</td>
			<td style="width:565px;">Concentration (mmol) 14.5</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Collangenase Solution 1</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Isolation Solution (30mL)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Bovine Fetal Albumin (BSA solution 5 ml)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;">Concentration (mmol) 1.67 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Collagenase Type 2</td>
			<td style="width:131px;">Worthington</td>
			<td style="width:125px;">LS004177</td>
			<td style="width:565px;">Concentration (mmol) 1 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Protease</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P6911</td>
			<td style="width:565px;">Concentration (mmol) 0.1 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;">Concentration (mmol) 0.05 mM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Collangenase Solution 2</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Isolation Solution (20mL)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Bovine Fetal Albumin (BSA solution 3.3 mL)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;">Concentration (mmol) 1.67 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Collagenase Type 2</td>
			<td style="width:131px;">Worthington</td>
			<td style="width:125px;">LS004177</td>
			<td style="width:565px;">Concentration (mmol) 1 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;">Concentration (mmol) 0.05 mM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">BSA solution</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Isolation Solution (40mL)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Bovine Fetal Albumin</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">A7906</td>
			<td style="width:565px;">Concentration (mmol) 400 mg</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;">Concentration (mmol) 1mM</td>
		</tr>
	</tbody>
</table>

assessment of myofilament ca2+ sensitivity underlying cardiac excitation-contraction coupling

心不全や不整脈は、世界中の死亡率および罹患率の主要な原因です。しかし、疾患のある心臓における病因および心筋機能不全のメカニズムは完全には明らかにされないままです。最近の説得力のある証拠は、筋フィラメントの変化は、Ca 2+感度心筋細胞における細胞内Ca 2+恒常性およびイオンチャネルの活動に影響を及ぼしていること健康と病気の心の中で心臓の活動電位と収縮を担う不可欠なメカニズムを示しています。実際、心臓の活動電位の基礎となるイオンチャネルやトランスポーターの活動（ 例えば Na +、 の Ca 2+およびK +チャネルおよびNa + -Ca 2+交換体）と細胞内Ca 2+のタンパク質（ 例えば 、リアノジン受容体およびCaの取り扱い筋小胞体（SERCA2aの）またはホスホランバンおよびそのリン酸化）2+ -ATPaseは、従来evaluするために測定されます心臓の興奮収縮（EC）カップリングの基本的なメカニズムを食べました。筋フィラメントが収縮と弛緩の機能単位であり、筋フィラメントの Ca 2+感受性が筋原繊維性能の実装では不可欠であるのに対し、膜と細胞内Ca 2+変化の両方の電気的活動は、ECカップリングのトリガ信号です。それが研究の焦点でない限り、それにもかかわらず、いくつかの研究では、心筋の機能解析に筋フィラメントのCa 2+感受性を組み込みます。ここでは、サルコメア短縮/再延長とのFura-2 AM（レシオメトリック検出）およびラット心臓から心筋細胞における筋フィラメントのCa 2+感受性の変化を評価を使用して、細胞内Ca 2+レベルを測定プロトコルを記述します。主な目的は、筋フィラメントのCa 2+感受性が機構的な分析のためのECカップリングに考慮されるべきであることを強調することです。私の総合的な調査翻訳および治療 ​​的価値のより効果的な戦略を設計するための貴重な情報を提供する健康と病気の心の中で筋細胞の収縮性を根底チャネル、イオントランスポーター、細胞内Ca 2+の取り扱い、および筋フィラメントの Ca 2+感受性に。

LV筋細胞は、正常と高血圧ラット心臓から単離されます。フィールドの刺激に応答して、明確な条線（サルコメアを表す）、安定した収縮との棒状筋細胞は、最適な筋細胞であると考えられていると録音（ 図2A）のために選択されています。 Fのigure 2Aに示す例では、フラ2 AMは、LV筋細胞が水平に配置され、筋細胞、記録フィールドの大部分?...

Watch this Scientific Journal Video about Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling at JoVE.com

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts.

筋フィラメントのCaの評価<sup&gt; 2+</sup&gt;感度基礎心臓興奮収縮連関

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial ...

assessment-myofilament-ca2-sensitivity-underlying-cardiac-excitation

Research

JoVE Journal

Biology

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

9.4K ビュー.  Seoul National University College of Medicine. This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts. 

ビデオ: Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

トカゲの運動の速度と方向の視覚感度のテスト

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

生物学

Investigating the Immunological Mechanisms Underlying Organ Transplant Rejection

免疫学的メカニズム深部臓器移植の拒絶反応の調査

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

焦点のCa 2 +</sup平滑筋の>過渡検出

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

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 ...

Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis.
The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture.
This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.

Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry.

サポニン透過性心臓繊維の呼吸計測の酸化的リン酸化の評価

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

血管のCaの可視化 2 +パラクリン派生ROSをきっかけにシグナリング

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

末梢血幹細胞移動の解析のためのマウス精巣挙筋で微小循環の生体顕微鏡

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

ダイナミックカルシウムの自動分析<sup&gt; 2 +</supイメージシーケンス内の&gt;の信号

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

時空のCaを測定する<sup&gt; 2 +</supシロイヌナズナ植物における&gt;シグナル

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

イメージングローカルCA<sup&gt; 2+</sup&gt;培養哺乳動物細胞内シグナル

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...