著者らは、顕微鏡実験で優れた技術支援のためにグレン·スローカム（ウィスコンシン医科大学）とコリーンA.ラビン（ニコンインスツルメンツ社）を感謝したいと思います。グレゴリー·ブラスは、原稿の重要な校正のために認められています。この研究は、（DVIに）米国腎臓学会から（ASに）1-15-BS-172を付与し、ベン·J·リップス研究員国立衛生研究所の助成金HL108880と米国糖尿病協会によってサポートされていました。

動物の使用と福祉制度動物実験委員会（IACUC）によってレビューされ、承認されたプロトコル以下の実験動物の管理と使用に関するNI​​Hガイドに付着する必要があります。 1.腎臓フラッシュ<ol><li> （ただし、異なる年齢や性別の他の株を適切に変更して使用することができ、スプラーグドーリー株である示唆）8から12週齢の雄ラットを使用してください。 </li><li> IACUCプロトコルによって許可された手順に従って、動物を麻酔。麻酔深度を監視し、動物を検査します。手術の詳細な説明は、1.3で実行される- 1.8 Ilatovskaya らに記載されています7。 </li><li>適切な麻酔の後、（長さ3インチまで）、腹部の正中切開を行い、温度制御手術台の上に動物を置き、大静脈と大動脈を発見。 </li><li>腹腔と腸間膜動脈と腹部周りに結紮糸を挿入これらの上記の大動脈。連結しないでください。 </li><li>腎動脈下の腹部大動脈を分析し、その周りに2合字を配置したが、合字の上に大動脈をクランプし、下糸を結ぶその後、​​連結していない鈍いです。 </li><li>クランプ以下（PBSを充填したシリンジポンプに取り付けられた）ポリエチレンPE50チューブを大動脈カテーテルを挿入し、第二の結紮糸でカテーテルを固定します。 、クランプを取り外し、ポンプをオンにし、腹腔動脈と大動脈および腸間膜を連結。すぐに圧力を緩和するために、腎静脈に切開します。 </li><li> /分6ミリリットルの速度で、2または3分間予備冷却PBSで大動脈を注入。 </li><li>灌流、消費税を停止し、7腎臓をデカプセル化し、PBS溶液中に氷の上に置きます。 IACUCによって承認されたプロトコルに従って、動物を安楽死させます。 </li></ol>ラット糸球体の2の単離<ol><li> RPMI 1640中の5％BSAの新鮮な溶液30mlを準備します。 </li><li>カミソリの刃やはさみを使用して、両方の腎臓の皮質を単離し、その後、均一になるまでミンチ。この手順は、以前に記載されている7。 </li><li>スパチュラを用いて（5％BSA / RPMI溶液に予備浸漬）100メッシュのステンレス鋼篩を通して、前のステップの間に細分化した組織を押してください。フロースルー及び重力によって収集し、フロースルーを140メッシュのふるいを通過することを可能にします。 </li><li> 、フロースルー予め浸漬200メッシュの篩を用いて、140メッシュのふるいから収集をフィルタろ液を捨て、そして10でふるいの上部を洗っ - で沈降糸球体を収集するために準備されたBSA / RPMI溶液15mlふるいです。 </li><li>最大20分間、チューブの底に糸球体の堆積物を15mlチューブに氷の上で糸球体を含むBSA / RPMI液を入れてみましょう。チューブの底に糸球体の濃縮が明らかに見られます。チューブ内の約2ミリリットルを残し、余分な溶液を除去します。 </li></ol> 3.シングルチャネルパッチクランプエルectrophysiology <ol><li> MW7万でそれらをコーティングすることにより、5×5ミリメートルのカバーガラスチップを準備 - 15万ポリʟリジン、および乾燥させてください。カバーガラスあたりの水に0.01％の滅菌濾過した溶液を約30μLを使用してください。 </li><li> RTへの実験的なソリューションをウォームアップし、パッチクランプ室とピペットを埋めます。 TRPCチャネルの監視の場合、mMの中で、浴溶液を使用します。126のNaCl、1のCaCl 2、10 HEPES、2のMgCl 2、10グルコース、pHが7.4;ピペット：126のNaCl、1.5のCaCl 2、10 HEPES、10グルコース; pHは7.4。 <ol><li>阻害すること（10mMのTEA、チャンネル） - 100μMのCaを遮断するニフルム酸またはDIDS（2+活性化Clで：推奨される（内因性チャネルの活性をブロックするためにピペット溶液に阻害剤を追加し、研究に関連されていません大コンダクタンスの Ca 2+ -依存性K +チャネル ）、10 nmのイベリオトキシン（のCa 2+活性化K +チャネルを遮断するために）、10μMのニカルジピン（N型Caをブロックします直接パッチクランプ実験前2+チャネル））。 </li></ol></li><li>静かに糸球体を含む溶液を混合し、次いでポリʟリジンコートカバーガラスチップに約50μLを適用します。糸球体は、約5分間添付してみましょう。 </li><li>浴溶液で予め充填されたパッチクランプ室に糸球体のガラスチップを移動します。付着していない糸球体の除去を確実にするために1分間、3ml /分の速度で室を灌流。 </li><li>セル接続モード7で、従来のパッチクランプ実験を行います。ガラスピペットを用いて（7から10MΩピペット抵抗）に、単離された糸球体の足細胞表面上に取り付けられたピペットは、 図2に示されている（穏やかな吸引を適用することにより、ピペットと足細胞膜との間に高抵抗シールを形成左）。 </li><li>セル付測定のために、8極ベッセルフィルタにより300 Hzの電流を低域通過。 </li><li> U6時間 - SEは最大4のパッチクランプ実験で糸球体を単離しました。氷の上で在庫糸球体の割合を保ちます。 </li></ol>足細胞の細胞内カルシウム濃度の4レシオメトリック共焦点蛍光測定<ol><li>コニカルチューブ0.5mlの（2.5で説明した）糸球体画分の500μLを置き、カルシウム色素のFuraレッド、AMとのFluo-4、AMを追加します。 2 mMのと（DMSOに溶解し、-20℃で保存）のFuraレッド、AMとのFluo-4、AM、それぞれの1 mMストック濃度を使用し、糸球体画分の500μlの各色素の2.5μLを使用します。直ちに染料の添加後、アルミニウムホイルでチューブを覆います。 </li><li> RTで1時間に少なくとも20分までのための回転シェーカー上に置いチューブ。 注：薬理学的薬剤は、この工程の間に加えることができます。 </li><li> 、カバーガラスを準備するポリʟリジンでそれらをカバーし、70℃に加熱されたプレートのセットを使用して乾燥を可能にします。 </li><li>カルシウム色素のロードはのcompletたらeは、ポリʟリジンコートカバースリップの解決策を含む糸球体の100μLを適用し、それらを約5分間、表面に固執しましょう​​。撮影室に糸球体付きカバースリップをマウントし、（（さ）mmで含む：145のNaCl、4.5のKCl、2のMgCl 2、10 HEPES、pHは7.35）浴溶液で灌流除去するために3ミリリットル/分の速度で付着していない糸球体、残りの染料。 </li><li> 488 nmの励起波長および発光フィルタ（それぞれのFluo-4及びフラ赤25分の525と25分の650ナノメートル）の共焦点レーザー走査顕微鏡を設定します。所望の周波数と解像度にイメージングソフトウェアを設定します。 </li><li>明視野で糸球体を検索し、その後、蛍光シグナルの検出をオンにします。信号の飽和を回避するために、各色素のためのレーザーの強さを調整します。直接ガラスに取り付けられている足細胞で焦点面を選択します。これは、薬物に応じて糸球体の収縮による影響を最小限に抑えますアプリケーション。希望の糸球体はよくガラスに接続されていることをダブルチェックしてください。 </li><li> 、選択の焦点面を撮像開始（高速のCa 2+過渡変化を可視化するために4秒に設定された周波数で512×512の画像を取る。高解像度画像のための60X / NA 1.4または類似の対物レンズを使用して）、関心のある薬物を適用および応答を記録します。 <ol><li> （糸球体の表面に足細胞のイメージングを確保するため、可能な限りガラスの表面に近い）は、所望の焦点面を選択します。 FLUO4とFuraRedチャネル上の蛍光の強度を確認し、糸球体は明らかに明視野で見ていることを確認してください。 </li><li>撮影を開始します。いずれかの薬剤の適用前に、レコードベースライン蛍光の少なくとも1分信号が安定していることを確認するために、（そこには突然のスパイクがされていないか、信号のフェージング）。 </li><li>マイクロピペットの助けを借りて、所望の薬を適用します。注意して、薬剤がよく拡散し、glomerに到達することができました確認してくださいウルス。必要に応じて静かに浴溶液を混合し、選択された焦点面を監視し、それがために薬物適用の焦点の外に移動しなかったことを確認してください。 </li><li> FLUO4とFuraRed信号用の蛍光強度の変化を記録します。記録信号がプラトーレベルに達するか、ピークに達し、その後ベースラインに戻るまで待機することにより、十分な長さであることを確認してください。必要に応じて任意の他の薬剤の溶液の変更や追加を行います。 </li><li>記録を停止し、ソフトウェアのネイティブ形式でファイルを保存します。 </li></ol></li></ol>カルシウム測定5.画像解析<ol><li>ネイティブND2形式で画像をインポートすることができNDユーティリティのプラグインを搭載したImageJのオープンソフトウェアで画像解析を実行します。 </li><li>イメージシーケンスをインポートします。チャンネルを分割し、hyperstackのグレースケールモードを使用するようにしてください。 </li><li> OへのImageJソフトウェアで分析→ツール→ROIマネージャパスに従ってくださいペンROIマネージャウィンドウ。 ROI Managerの楕円形選択ツール、「追加（T）」関数を使用して、興味のあるいくつかの領域（足細胞）を選択します。最後のROIとしては、バックグラウンドでエリアを選択します。 （保存→詳細）選択したROIを保存します。 </li><li>分析のために選択されたチャンネルを含むウィンドウを強調表示します。 、ROI Managerでマルチ測定機能→詳細を使用して飛び出すの対話で「1行あたりのスライス」のボックスに印を付け、[OK]をクリックします。結果は結果ウィンドウで設定可能な形式で表示されます。→設定測定メニューオプションの結果に入り、「グレー値の平均」と各ROIのための画素強度値を計算選択し、で表示されます各ROIのための別々の列に結果ウィンドウ。 </li><li>好適なデータ解析ソフトウェアに各チャネルの測定されたROIの輝度値（のFluo-4とのFuraレッド）をコピーします。各DATからのバックグラウンド強度値を減算しますポイント。 </li><li>各時点でのFuraレッドチャネルへのFluo-4の強度の比を計算します。各ROIのためのCa 2+トランジェントのプロット散布/ラインポイント時間の変更。選択された糸球体の平均/ SE値を計算します。 注：時間列は4.5で選択した撮影頻度に応じて設定する必要があります。 </li></ol>フルオ4蛍光シグナルを使用した6細胞内カルシウム濃度の計算<ol><li>糸球体のサンプルを取り、4.7のステップに実験プロトコルを実行します。バックグラウンド蛍光を記録した後に浴溶液に、レコード蛍光強度の増加（10μMであるべき浴室中の最終濃度）イオノマイシンを加えます。強度が最大に達し、崩壊が始まると、蛍光8をクエンチする（最終濃度が5 mMのでなければならない）のMnCl 2を追加します。 </li><li> 6.1で得られたデータを分析します。従っのFluo-4 ROI信号強度値が得られるコピー好適な解析ソフトウェアによって5.1から5.5に記載されているプロトコルに。 </li><li>式を使用して、ROIに対応する足細胞で（NMで）細胞内カルシウム濃度を計算します。 <img alt="式（1）" src="/files/ftp_upload/52850/52850equation1.jpg" /> K dはフルオ4（345 nM）をするための一定の予め決め解離であり、Fは、あなたが（ベースライン）のためのカルシウム濃度を計算している時点での強度であり、F min及びF maxはでの強度値であり、 （イオノマイシン適用後）と（のMnCl 2を有する）蛍光の消光後の最大カルシウム負荷の点、それぞれ（ 図3参照 ）。 </li></ol>

<ol>
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Commun. 408, 242-247 (2011).</li><li>Peti-Peterdi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+wave+of+tubuloglomerular+feedback.">Calcium wave of tubuloglomerular feedback.</a> Am. J. Physiol. Renal Physiol. 291, F473-F480 (2006).</li><li>Peti-Peterdi, J., Warnock, D. G., Bell, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+II+directly+stimulates+ENaC+activity+in+the+cortical+collecting+duct+via+AT(1)+receptors.">Angiotensin II directly stimulates ENaC activity in the cortical collecting duct via AT(1) receptors.</a> J. Am. Soc. Nephrol. 13, 1131-1135 (2002).</li><li>Ilatovskaya, D. V., Palygin, O., Levchenko, V., Staruschenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+characterization+of+the+P2+receptors+profile+in+the+podocytes+of+the+freshly+isolated+rat+glomeruli.">Pharmacological characterization of the P2 receptors profile in the podocytes of the freshly isolated rat glomeruli.</a> Am. J. Physiol. Cell Physiol. 305, C1050-C1059 (2013).</li><li>Ilatovskaya, D. V., 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=Angiotensin+II+has+acute+effects+on+TRPC6+channels+in+podocytes+of+freshly+isolated+glomeruli.">Angiotensin II has acute effects on TRPC6 channels in podocytes of freshly isolated glomeruli.</a> Kidney Int. 305, C1050-C1059 (2014).</li><li>Schaldecker, 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=Inhibition+of+the+TRPC5+ion+channel+protects+the+kidney+filter.">Inhibition of the TRPC5 ion channel protects the kidney filter.</a> J. Clin. Invest. 123, 5298-5309 (2013).</li><li>Roshanravan, H., Dryer, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+acting+through+P2Y+receptors+causes+activation+of+podocyte+TRPC6+channels:+role+of+podocin+and+reactive+oxygen+species.">ATP acting through P2Y receptors causes activation of podocyte TRPC6 channels: role of podocin and reactive oxygen species.</a> Am. J. Physiol. Renal Physiol. 306, F1088-F1097 (2014).</li><li>Anderson, M., Roshanravan, H., Khine, J., Dryer, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+II+activation+of+TRPC6+channels+in+rat+podocytes+requires+generation+of+reactive+oxygen+species.">Angiotensin II activation of TRPC6 channels in rat podocytes requires generation of reactive oxygen species.</a> J. Cell. Physiol. 229, 434-442 (2014).</li></ol>

著者らは、開示することは何もありません。

ここで説明する手法は、げっ歯類の糸球体の足細胞によるカルシウム処理の分析を可能にします。この技術は、パッチクランプ単一チャンネル電気生理学、蛍光レシオメトリック共焦点イメージングの適用を可能にします。しかしながら、両方のアプローチは、それ自体で、別々に使用することができます。提案されたプロトコルは、1）腎フラッシュなど、いくつかの比較的単純なステップ...

腎臓は、様々な物質のための恒常性バランスを維持し、総血圧を決定するようにして血液量を調節します。ハイパーまたは低血圧に至るまでに腎臓濾過、再吸収や分泌リードの障害または付随する病理学的状態は、最終的に腎臓移植を必要とする末期腎疾患を終了します。毛細血管内皮、基底膜および上皮細胞の単電池層- -スリットダイアフラムの完全性と機能1の維持に重要な役割を果たし足細胞、腎フィルタリング部（糸球体）は、3つの層からなります。選択透過性糸球体フィルタにおける機能不全は、蛋白尿などの巨大分子の尿損失の原因となります。種々の薬剤は、糸球体濾過障壁の完全性を決定し、それらの足細胞足突起の構造に影響を及ぼし得ます。 足細胞は、逮捕されるの維持に関与していますeruli濾過機能。これは、足細胞による不適切なカルシウム処理は、細胞損傷につながり、腎2,3の様々な形の進行に重要な役割を果たしていることが確立されています。したがって、細胞内カルシウム濃度の変化の直接測定を可能にするモデルの開発は、足細胞の機能の研究のための手段になります。単離された糸球体は、以前アルブミン反射係数の測定4および全細胞電気生理学的パッチクランプ測定5,6の積分携帯電流の評価を変更するなど、多くの研究で使用しました。本論文では、研究者は、薬剤の用途に応じて、細胞内カルシウム濃度の変化を測定し、細胞内カルシウムの基礎レベルを推定し、個々のカルシウムチャネルの活性を評価することを可能にするプロトコルを記述します。 Ratometricカルシウム濃度の測定値とパッチクランプelectrophysiologyはそれぞれ、足細胞及びチャネル内の活性細胞内カルシウム濃度の変化を決定するために使用しました。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Fluo4 AM</td>
			<td>Life Technologies</td>
			<td>F14217</td>
			<td>500&#181;l in DMSO</td>
		</tr>
		<tr>
			<td>FuraRed AM</td>
			<td>Life Technologies</td>
			<td>F-3020</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic acid</td>
			<td>Sigma-Aldrich</td>
			<td>F-68&#160;</td>
			<td>solution</td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma-Aldrich</td>
			<td>I3909-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube rotator</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-090-753</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon confocal microscope (inverted)</td>
			<td>Nikon</td>
			<td>Nikon A1R</td>
			<td>&#160;Laser exitation 488nm. Emission filters 500-550nm and 570-620nm</td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Nikon</td>
			<td>Plan Apo 60x/NA 1.4 Oil</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glass</td>
			<td>Thermo Scientific</td>
			<td>6661B52</td>
			<td></td>
		</tr>
		<tr>
			<td>High vacuum grease</td>
			<td>Dow Corning</td>
			<td></td>
			<td>Silicone Compound</td>
		</tr>
		<tr>
			<td>Software</td>
			<td>Nikon</td>
			<td>Nikon NIS-Elements&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Recording/perfusion chamber</td>
			<td>Warner Instruments</td>
			<td>RC-26</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Patch Clamp amplifier</td>
			<td>Molecular Devices</td>
			<td>MultiClamp 700B</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquisition System</td>
			<td>Molecular Devices</td>
			<td>Digidata 1440A</td>
			<td>Axon Digidata&#174; System</td>
		</tr>
		<tr>
			<td>Low Pass Filter</td>
			<td>Warner Instruments</td>
			<td>LPF-8</td>
			<td>8 pole Bessel</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B150F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instrument Co</td>
			<td>P-97</td>
			<td>Flaming/Brown type micropipette puller</td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige</td>
			<td>MF-830</td>
			<td>Japan</td>
		</tr>
		<tr>
			<td>Motorized Micromanipulator</td>
			<td>Sutter Instrument Co</td>
			<td>MP-225</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvibration isolation table</td>
			<td>TMC</td>
			<td></td>
			<td>equipped with Faraday cage</td>
		</tr>
		<tr>
			<td>Multichannel valve perfusion system</td>
			<td>AutoMake Scientific</td>
			<td>Valve Bank II</td>
			<td></td>
		</tr>
		<tr>
			<td>Recording/perfusion chamber</td>
			<td>Warner Instruments</td>
			<td>RC-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td>Molecular Devices</td>
			<td>pClamp 10 . 2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicardipine</td>
			<td>Sigma-Aldrich</td>
			<td>N7510</td>
			<td></td>
		</tr>
		<tr>
			<td>Iberiotoxin</td>
			<td>Sigma</td>
			<td>I5904-5UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Niflumic acid</td>
			<td>Sigma-Aldrich</td>
			<td>N0630</td>
			<td></td>
		</tr>
		<tr>
			<td>DIDS</td>
			<td>Sigma-Aldrich</td>
			<td>D3514-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>TEA chloride</td>
			<td>Tocris</td>
			<td>T2265</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Life Technologies</td>
			<td>11835030</td>
			<td>without antibiotics</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A8327</td>
			<td>30% albumin solution</td>
		</tr>
		<tr>
			<td>Temperature controlled surgical table&#160;</td>
			<td>MCW core</td>
			<td></td>
			<td>for rodents</td>
		</tr>
		<tr>
			<td>Steel sieves:</td>
			<td></td>
			<td></td>
			<td>#100 (150 &#956;m), 140 (106 &#956;m)</td>
		</tr>
		<tr>
			<td>Gilson, Inc&#160; SIEVE 3 SS FH NO200</td>
			<td>Fisher Sci</td>
			<td>50-871-316</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilson, Inc&#160; SIEVE 3 SS FH NO270</td>
			<td>Fisher Sci</td>
			<td>50-871-318</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilson, Inc&#160; SIEVE 3 SS FH NO400</td>
			<td>Fisher Sci</td>
			<td>50-871-320</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;mesh 200&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>s4145</td>
			<td>screen for CD-1</td>
		</tr>
		<tr>
			<td>Binocular microscope</td>
			<td>Nikon</td>
			<td>Eclipse TS100</td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular microscope</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump-based perfusion system</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>polyethylene tubing</td>
			<td>Sigma-Aldrich</td>
			<td>PE50</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluorane anesthesia</td>
			<td><a href="http://www.vetequip.com/">http://www.vetequip.com/</a></td>
			<td>911103</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Other basic reagents</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

single-channel analysis and calcium imaging in the podocytes of the freshly isolated glomeruli

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

ここでは、足細胞内カルシウム濃度の急激な変化を測定するという問題に取り組んだ。 図1は、新しくの足細胞での高解像度ライブ蛍光共焦点イメージングおよび単一のイオンチャネル活性の記録を行うために設計された実験プロトコルの概略図を示します分離されたげっ歯類糸球体。ラットを麻酔した後に簡単に説明すると、腎臓は血液のそれらをクリアするためにPBSでフラッ...

Watch this Scientific Journal Video about Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli at JoVE.com

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

新たに単離した糸球体の足細胞における単一チャネル分析とカルシウムイメージング

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...

single-channel-analysis-calcium-imaging-podocytes-freshly-isolated

Research

JoVE Journal

Biology

10.9K ビュー.  Medical College of Wisconsin. Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli. 

ビデオ: Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

FDG micro-PET/CTマウスにおける褐色脂肪の機能イメージング

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

生物学

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

アポトーシス細胞のクリアランスのイメージング中に生きる<em&gt;ショウジョウバエ</em&gt;胚

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

無傷骨格筋線維内のカルシウムスパークスの評価

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

単離された血液灌流ラットの肺の準備にシングル微小血管透過性を定量化する

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

浸透水透過係数（Pの測定<sub&gt; F</sub&gt;）球状細胞の：例として単離された植物プロトプラスト

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

新たに単離したPKD上皮の機能的特性を監視するためにパッチクランプし、ライブ蛍光顕微鏡を実装

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

リビングヒト臍帯静脈内皮細胞における高解像度タイムラプスイメージングと微小管ダイナミクスの自動解析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

マウス単離された灌流腎臓テクニック

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

ゼブラフィッシュ ランゲルハンス島における単一セルの解像度カルシウム イメージングを用いた β-細胞機能の解析

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

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 ...