私たちは、施設やイオンチャネルの研究のための優れた環境をご提供するための生理学と膜生物学、カリフォルニア大学デ​​ービス校の学科に感謝しています。

1。一休みするヒトTリンパ球の調製<ol><li> RosetteSepヒトT細胞の濃縮カクテルとRosetteSep密度培地を用いて、製造元の指示に従って、人間の血液サンプルからT細胞を精製する。結果として得られる細胞集団は95％CD3 +安静時のT細胞が含まれている必要があります。私たちは、カリフォルニア大学デ​​ービス校の内部審査委員会によって承認されたプロトコールに従って健康なボランティアから採取した末梢血サンプルからのヒトTリンパ球を精製する。 </li><li>単離後、10％ウシ胎児血清、2％グルタミン- I、1％RPMI 1640を添加した、グルタミンおよびHEPESを含むRPMI - 1640培地を含む細胞培養培地中で0.5 × 10 6細胞/ mLの密度で休んでT細胞を再懸濁ビタミンソリューション、1％RPMI 1640アミノ酸液、1％ピルビン酸ナトリウム、および0.03％β-メルカプトエタノール。 </li><li>場所細胞の細胞培養フラスコにサスペンション、°加湿インキュベーター中、5％CO 2で、最大24時間の前に実験に、37で維持する。 </li></ol> 2。記録チャンバーの準備<ol><li>以下は、録音室を準備するために必要とされる。）シリコンO -リング、b）のラウンドは、25mmの製造元の指示に従って調製Sylgard184を混ぜ、70％エタノールおよび蒸留H 2 Oで洗浄カバーグラス、C）、、およびd） 2つの60 X 15ミリメートルのペトリ皿。 </li><li>場所〜60x15 mmのシャーレに混合Sylgard184 0.5 mLの。 </li><li> Sylgard184使用して鉗子にO -リングの底部を浸します。 </li><li>このような別のペトリ皿のような清浄表面上にO -リングを配置することにより、O -リングの縁から過剰Sylgard184を削除します。 </li><li>カバースリップの上にリングを置き、それを押してください。 </li><li> 85℃40〜60分間、またはSylgard184まで、Cが重合されている時にチャンバーを加熱することによってSylgard184を治す。 </li><li>無塵容器に録音室に保管してください。 </li></ol> 3。パッチピペットの準備<ol><li>実験日に、ガラスキャピラリーやピペットプラーを使用して〜2μmの先端径でピペットを引き出します。私たちは、フィラメント（：1.5 mmとID：1.10 mm外径）とホウケイ酸塩チューブを使用してください。ほこりのない場所で保管してピペット。 </li><li>標準的な手順9に従ってSylgard184またはHIPEC R6101の半導体保護コーティング、とコートは、ピペットチップ。 </li><li>静かに実験前にピペットを火 - ポーランド語。 </li></ol> 4。パッチクランプのセットアップの準備<ol><li>あなたのパッチクランプアンプを制御するソフトウェアでgigaseal形成のためのマクロを設定し、保存します。我々は、Windows XPやデータ取得用のパルスソフトウェアでサポートされているEPC - 10アンプを使用してください。我々は、EPC - 10マニュアルによると、&quot;オンセル&quot;、および&quot;細胞全体の&quot;マクロ&quot;をセットアップ&quot;を設定し、すべての実験のためにそれらを使用してください。 </li><li>前の実験にお風呂のソリューションと、表1に記載されているピペットのソリューションを用意し、最大1週間は4℃で保管します。 </li></ol><table border="1"><tbody><tr><td>化学物質</td><td colspan="3">バスソリューション（MM） </td><td>ピペットソリューションズ（MM） </td></tr><tr><td></td><td>第1位</td><td> ＃2 </td><td>第3位</td><td></td></tr></tbody><tbody><tr><td> CH 3 SO 3のNa </td><td> 130 </td><td> 110 </td><td> 125 </td><td> - </td></tr><tr><td> NaClの</td><td> 2 </td><td> 4 </td><td> 5 </td><td> - </td></tr><tr><td>のCa（OH）2 </td><td> - </td><td> 20 </td><td> - </td><td> - </td></tr><tr><td> MgCl 2の </td><td> 3 </td><td> 1 </td><td> - </td><td> 5 </td></tr><tr><td> MgSO 4を </td><td> - </td><td> - </td><td> - </td><td> 2 </td></tr><tr><td> HEPES </td><td> 10 </td><td> 10 </td><td> 10 </td><td> 15 </td></tr><tr><td> HEDTA </td><td> - </td><td></td><td> 10 </td><td> - </td></tr><tr><td> EDTA </td><td> - </td><td> - </td><td> 1 </td><td> - </td></tr><tr><td> CS -spartate </td><td> - </td><td> - </td><td> - </td><td> 125 </td></tr><tr><td> BAPTA </td><td> - </td><td> - </td><td> - </td><td> 12 </td></tr><tr><td>グルコース</td><td> 10 </td><td> 10 </td><td> 10 </td><td> - </td></tr></tbody></table> 表1。全細胞CARC現在のレコーディングのためのソリューション。 すべての化学物質はSigma - Aldrich、セントルイス、MOから購入した。 <ol start="3"><li>パッチピペット溶液及び各浴溶液10との間の液体接合のポテンシャル（LJ）を決定します。あなたの集録ソフトウェアを使用して適切な場所にLJの値を保存します。例えば、パルスのプログラムで&quot;アンプ&quot;ウィンドウで&quot;LJ&quot;の制御にLJの値を挿入します。 </li><li>実験前に買収のソフトウェアの適切な場所で、図1に示すように、刺激プロトコルを、構成し、保存します。パルスのプログラムでは、我々は&quot;パルス生成&quot;ウィンドウで、買収のプロトコルを設定します。 <img src="/files/ftp_upload/2346/2364fig1.jpg" alt="図1" /> 図1。 2の（5 - - 0.5 Hz）の刺激のプロトコルは、-120から持続時間50ミリ秒の100 mVまでの電圧ランプはすべての0.2に適用されます。ランプの間に保持電位（V H）が 30 mVに保持されます。 </li></ol> 5。顕微鏡ステージ上に取り付けセル<ol><li>細胞を播種前にして〜20分と洗浄H 2 Oとし、ハンクス平衡塩溶液（HBSS）を持つためのポリ- L -リジンでコーティング録音室を。 </li><li> 37℃で20分間チャンバー内でインキュベート中に0.2〜0.5 × 10 6個の細胞を含む細胞懸濁液のプレート500μL℃に</li><li>チャンバーのホルダーに付着した細胞と録音室を確保する。我々は、ベースはカバースリップの上に対して上部軽く押したまま、カバーの底部をサポートするカスタムメイドのチャンバーのホルダーを、使用してください。 </li><li>倒立顕微鏡のステージ上のチャンバホルダーを置きます。 </li><li>透過光で細胞に焦点を当てる。我々は、40X油浸対物レンズを使用。 </li><li>浴中にAg / AgCl参照電極を置きます。 </li><li> multibarrel、重力駆動型の灌流システムを合わせます。流入管の先端が視野にカバースリップの近くに45 °の角度で配置する必要があります。 </li><li>流入管の先端に吸引管の​​反対側に置きます。 </li><li>でチャンバーを灌流リン酸緩衝生理食塩水（PBS）またはHBSSは、細胞培養培地とゆるく付着した細胞を洗い流す。 </li></ol> 6。 CRACチャネル電流の記録<ol><li>しっかりとカバースリップに接続されているセルを見つける。我々は一般的に滑らかな外表面を持つ平均的なサイズの、円形セルを選択します。 </li><li> 3mMのMg 2 +の含有浴溶液SERCAポンプをブロックするために8〜10分間0.5から1μMのタプシガルギンを含む第1位（表1）、+ -遊離Ca 2録音室を灌流。我々は、前のgigaseal形成へのストアを枯渇させるために、この手順を実行します。 </li><li>エッペンドルフmicroloaderとP - 20変位のピペットを使用して、ピペット溶液（表1）でパッチピペットを記入し、アンプのヘッドステージ上にピペットホルダーにパッチピペットを挿入する。 のCa 2 +キレート剤BAPTAは、受動的に店舗を破壊すると+ -依存性のCRACチャネルの不活性化のCa 2を減らすためにピペット溶液に含まれています。 </li><li>パッチピペット内の風呂に入る前に、正圧（H 2 Oの〜3インチ）の少量を適用します。我々は、パッチピペット内部に、正または負圧を作成するための圧縮空気と真空ラインに接続されたカスタムメイドの圧力吸引装置を使用してください。 </li><li>顕微鏡を介して視覚的なコントロール下でバスにパッチピペットを下げます。 &quot;オシロスコープ&quot;ウィンドウでは、ピペットを流れる電流が表示されるはずです。電流振幅のステップ状の変化がプリセットマクロによって適用される小振幅の電圧パルス（試験パルス）によって誘発されるべきである。この時点では、ピペットの抵抗の値を決定することができるはずです。私たちのピペットの抵抗は、通常、MΩ5月6日です。 </li><li>ピペットと参照電極間に生成された&quot;オフセット&quot;の可能性を修正してください。 </li><li>それは細胞膜に触れるまで、顕微鏡を介して視覚的制御の下で、近い細胞にピペットの先端をもたらす。 </li><li>パッチピペット内部の負圧（H 2 Oの20〜40インチ）を適用します。 </li><li> &quot;オシロスコープ&quot;ウィンドウでgigaseal形成を監視します。あなたは、テストパルス、減少し、&gt; 5GΩにピペット抵抗の値が増加することによって誘導される電流の振幅を、表示されます。 FE以内gigasealフォームwの数秒が、それは安定したgigasealを達成するために1〜2分かかる場合があります。 </li><li>ピペット容量（&quot;C -ファースト&quot;）を補償する。 </li><li> 20ccの注射器を使用して追加の負圧を適用してパッチ膜を破る。 </li><li> 30 mVに保持電位を設定します。正の保持電位は、カルシウム依存CRACチャネルの不活性化を防ぐことができます。 </li><li>細胞の膜容量（&quot;C -遅い&quot;）を補償C -遅いの値を保存または記録する。 </li><li>アクセス抵抗&quot;R s&quot;の値を確認してください。我々の実験では、R sは 、通常、MΩ70から20までです。 </li><li>のCa 2 0.5 Hzの周波数（図1）+ -フリー3mMのMg 2 +を含有浴溶液＃1の電圧ランプのシリーズを適用し始める。 Mg 2 +のためのCRACチャネルの低透過性のために&quot;リーク&quot;現在と比較してこのソリューションでは、CRAC電流は無視できるほど小さいです。 &quot;漏れ&quot;電流のように将来の分析で浴溶液​​＃1に記録されている現在のトレースを保存して使用してください。 -100 mVで&quot;リーク&quot;電流の絶対振幅は、以下または5 pAのと同じである必要があります。セルが&quot;漏れ&quot;の場合は、新しいパッチピペットと新しいセルを使用する場合に比べて実験してスタートを停止する。 </li><li> CRACチャネル（I のCa - CRAC）経由でのCa 2 +電流を記録するには、 潅流バルブを切り替えることで20mMのCa 2 +の含有浴溶液＃2（表1）でのCa 2 + -フリー3mMのMg 2 +を含有浴溶液＃1を交換してください。これは、Ca 2 +のCRACチャネルを経由してフローすることを可能にし、内向き電流を生成する。 &quot;オシロスコープ&quot;ウィンドウでは、内側に私のCa - CRAC（図2）整流の開発が表示されます。負電圧での電流の振幅は、CRAC電流のCa 2 +依存性増強のために約1分間増加していきます。 </li><li>一過性のNa + CRACチャネルを介して電流を記録するために必要な5 Hzの、の電圧ランプの刺激の頻度を増加させる。 </li><li> Na +の含有二価カチオンを含まない灌流バルブを切り替えることにより、ソリューション＃3（表1）と20mMのCa 2 +の含有浴溶液＃2を交換してください。 &quot;オシロスコープ&quot;ウィンドウでは、内向きのNa + CRACチャネル（;図2 I のNa - CRAC）を介して電流整流大きな振幅の過渡的な開発が表示されます。 </li><li>一つは、現在の&quot;リーク&quot;を記録するために実験の終了時に1から5μMラ3 +を含む20mMのCa 2 +含有浴溶液＃2を適用される場合があります。しかし、我々は時間をかけて&quot;リーク&quot;電流が変化するため、このアプローチが有用見つけることができませんでしたし、それは実験の終了時に大きくまたは小さくなる場合があります。私たちは、&quot;リ ​​ーク&quot;現在として実験の開始時に現在のCa 2で記録した+ -フリー3mMのMg 2 +を含有浴溶液＃1を取ることを好む。 </li><li>さらに分析するために記録された現在のトレースを保存します。 </li></ol> 7。データ解析<ol><li>解析ソフトウェアに保存されている現在のレコードを取得。我々は分析のためにパルスプログラムを使用してください。 </li><li>初めに、電圧ランプの終わりに現在の振幅を測定します。我々は、通常、-100 mVで、パルスプログラムの&quot;オシロスコープ&quot;ウィンドウ内のカーソルのペアを使用して100 mVでの電流振幅を測定する。 </li><li>さらに分析およびグラフィカルなプレゼンテーションのためのグラフィックプログラムに現在の振幅の値をエクスポートします。我々は、Origin科学グラフと解析ソフトウェア、バージョン7を使用してください。 </li></ol> 8。代表的な結果<img src="/files/ftp_upload/2346/2346fig2.jpg" alt="図2" /> 図2。安静時のヒトT細胞におけるCRAC電流。（）、-100 mVの（黒丸）と+100 mVの（白丸）でのホールセル電圧クランプ構成で記録されたCRAC電流の時間経過。 gigaseal形成に先立って、細胞を、Ca 2年〜10分間プレインキュベーションした+ -フリー3mMのMg 2 +を含有浴溶液、0.5μMのタプシガルギンを含む第1位。ブレークイン後、お風呂のソリューションは、順番に次のように適用されました：のCa 2 + -フリー3mMののMg 2のCa 2 +含有浴溶液＃2 20 mMのそれに続く+含有浴溶液＃1（0のCa + 3mg）を、 （20 Ca）の、浴溶液＃2（20 Ca）のに続いて二価カチオンを含まない浴溶液＃3（DVF）、続いて。セルは、図1に示すように電圧ランプのシリーズで刺激した。ランプの周波数は、浴溶液＃3（DVF）とすべての他のソリューションでは0.5 Hzで5Hzのだ。 20mMのCa 2 +の含有浴溶液＃2とDVFの浴溶液＃3の私のNa - CRACの高速過渡開発のアプリケーションの後に私のCa - CRACの遅い開発に注意してください。 （B）、代表、現在のトレースは、+ -フリー3mMのMg 2 +を含有浴溶液＃1（のCa 2に電圧ランプの間に記録さ&quot;リーク&quot;）、20mMのCa 2 +の含有浴溶液＃2（20 Ca）の、そして浴溶液＃3（DVF）。 （C、D）、私のCa - CRAC（C）と私のNa - CRAC（D）の電流-電圧の関係は、20mMの電圧ランプ中のCa 2 +含有お風呂に記録された電流から&quot;リーク&quot;電流を差し引いたパネル（B）に示すように解第2位（20 Ca）の、そして浴溶液＃3（DVF）。

<ol>
	<li>Parekh, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Store-operated+CRAC+channels:+function+in+health+and+disease.">Store-operated CRAC channels: function in health and disease.</a> Nat Rev Drug Discov. 9, 399-410 (2010).</li><li>Feske, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRAC+channelopathies.">CRAC channelopathies.</a> Pflugers Arch. , (2010).</li><li>Lewis, R. S., Cahalan, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogen-induced+oscillations+of+cytosolic+Ca2++and+transmembrane+Ca2++current+in+human+leukemic+T+cells.">Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells.</a> Cell Regul. 1, 99-112 (1989).</li><li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+inactivation+of+depletion-activated+calcium+current+(ICRAC)+due+to+local+calcium+feedback.">Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback.</a> J Gen Physiol. 105, 209-226 (1995).</li><li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+calcium-dependent+inactivation+of+depletion-activated+calcium+current.+Store-dependent+and+-independent+mechanisms.">Slow calcium-dependent inactivation of depletion-activated calcium current. Store-dependent and -independent mechanisms.</a> J Biol Chem. 270, 14445-1451 (1995).</li><li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-dependent+potentiation+of+store-operated+calcium+channels+in+T+lymphocytes.">Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes.</a> J Gen Physiol. 107, 597-610 (1996).</li><li>Prakriya, M., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+characterization+of+currents+through+store-operated+CRAC+channels+and+Mg2+-inhibited+cation+(MIC)+channels.">Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels.</a> J Gen Physiol. 119, 487-507 (2002).</li><li>Fomina, A. F., Fanger, C. M., Kozak, J. A., Cahalan, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+channel+properties+and+regulated+expression+of+Ca(2+)+release-activated+Ca(2+)+(CRAC)+channels+in+human+T+cells.">Single channel properties and regulated expression of Ca(2+) release-activated Ca(2+) (CRAC) channels in human T cells.</a> J Cell Biol. 150, 1435-1444 (2000).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-Channel Recording. , (1995).</li><li>Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+for+liquid+junction+potentials+in+patch+clamp+experiments.">Correction for liquid junction potentials in patch clamp experiments.</a> Methods Enzymol. 207, 123-1231 (1992).</li></ol>

利害の衝突は宣言されません。

これらの細胞中の内因性CRAC電流振幅が小さいセルサイズ（安静時のヒトT細胞の直径は5〜8μmの範囲にある）のために小さいため、ヒトT細胞の休止のCRAC電流の電気生理学的研究は困難な作業です。ここで、我々は確実に末梢血単核細胞から単離されたヒトTリンパ球を休んでCRAC電流を記録するためにステップバイステップの手順を示します。この技術は、私たちはより良い正常および病的な?...

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<td>RosetteSep Human T Cell Enrichment Cocktail</td>
<td>&nbsp;</td>
<td>StemCell Technologies, Vancouver, BC, Canada</td>
<td>15061</td>
<td>&nbsp;</td>
<tr>
<td>RosetteSep Density Medium</td>
<td>&nbsp;</td>
<td>StemCell Technologies </td>
<td>15705</td>
<td>&nbsp;</td>
<tr>
<td>RPMI-in 1640 medium w/glutamine/HEPES</td>
<td>&nbsp;</td>
<td>Fisher, Waltham, MA</td>
<td>SH3025501</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Calf Serum</td>
<td>&nbsp;</td>
<td>Omega Scientific, Tarzana, CA</td>
<td>FB-01</td>
<td>&nbsp;</td>
<tr>
<td>GlutaMAX-I (100X solution)</td>
<td>&nbsp;</td>
<td>Invitrogen, Carlsbad, CA</td>
<td>35050</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640 vitamin solution (100X)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>7256</td>
<td>&nbsp;</td>
<tr>
<td>1640 amino acids solution (50X)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>R7131</td>
<td>&nbsp;</td>
<tr>
<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8636</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-Mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M7522</td>
<td>&nbsp;</td>
<tr>
<td>Inositol trisphosphate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>19766</td>
<td>&nbsp;</td>
<tr>
<td>BAPTA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A4926</td>
<td>&nbsp;</td>
<tr>
<td>Poly-L-Lysine Hydrobromide</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P2636</td>
<td>&nbsp;</td>
<tr>
<td>Lanthanum Chloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>262072</td>
<td>&nbsp;</td>
<tr>
<td>Thapsigargin</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>586005</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 Silicon Elastomer Kit</td>
<td>&nbsp;</td>
<td>Dow Corning, Midland, MI</td>
<td>3097358-1004</td>
<td>&nbsp;</td>
<tr>
<td>HIPEC R6101 Semiconductor Protective Coating</td>
<td>&nbsp;</td>
<td>Dow Corning, Midland, MI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>63-500 Series High-Performance Vibration Isolation Lab Table</td>
<td>&nbsp;</td>
<td>Technical Manufacturing, Peabody, MA</td>
<td>63-540</td>
<td>&nbsp;</td>
<tr>
<td>EPC 10 patch clamp amplifier with headstage</td>
<td>&nbsp;</td>
<td>HEKA Instruments, Bellmore, NY</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator </td>
<td>&nbsp;</td>
<td>Sutter Instrument, Novato, CA </td>
<td>MP-285</td>
<td>&nbsp;</td>
<tr>
<td>Olympus 1X71 Inverted microscope with 40x oil immersion objective</td>
<td>&nbsp;</td>
<td>Olympus America, Center Valley, PA</td>
<td>1X71</td>
<td>&nbsp;</td>
<tr>
<td>Windows Computer</td>
<td>&nbsp;</td>
<td>Dell </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pulse software</td>
<td>&nbsp;</td>
<td>HEKA Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Origin Scientific Graphing and Analysis Software</td>
<td>&nbsp;</td>
<td>OriginLab, Northampton, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Patch pipette puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass with filament (O.D.: 1.5mm and I.D.: 1.10mm</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>BF150-110-7.5</td>
<td>&nbsp;</td>
<tr>
<td>Narashige's Microforge</td>
<td>&nbsp;</td>
<td>Tritech Research, Los Angeles, CA</td>
<td>MF-830</td>
<td>&nbsp;</td>
<tr>
<td>Silicon O-rings </td>
<td>&nbsp;</td>
<td>McMASTER-CARR, Santa Fe Springs, CA</td>
<td>111 S70</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips 25 mm </td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>12-545-102 25 mm 25CIR.-1</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

whole-cell recording of calcium release-activated calcium (crac) currents in human t lymphocytes

Tリンパ球で、細胞内Ca 2 +ストアからのCa 2 +枯渇は、カルシウムのリリース-活性化カルシウム（CRAC）チャネルと呼ばれる細胞膜のCa 2 +チャネルの活性化につながる。 CRACチャネルは、T細胞の増殖と遺伝子発現の調節において重要な役割を果たしている。 T細胞の異常なCRACチャネル機能は、重症複合免疫不全や自己免疫疾患1、2にリンクされています。ヒトT細胞のCRACチャネルの機能を研究することは正常な免疫反応を調節する新たな分子メカニズムを明らかにし、関連するヒト疾患の原因を解明することができる。膜電流の電気生理学的記録は、機能的なチャネルのプロパティとその規制の最も正確な評価を行うこと。 Jurkat T細胞、ヒト白血病T細胞株、のCRACチャネル電流の電気生理学的評価が最初に20年以上前に3を実行された、しかし、正常なヒトT細胞におけるCRAC電流測定は困難な課題である。正常T細胞でCRACチャネル電流を記録するの難しさは、血液由来のTリンパ球は、Jurkat T細胞よりもはるかに小さいサイズと、従って、内因性の細胞全体のCRAC電流は振幅が非常に低いものであるという事実によって悪化されています。ここで、我々は日常的に健康なボランティアの末梢血から単離されたヒトT細胞の休止のCRACチャネル経由でのCa 2 +又はNa +電流を記録するために使用するステップバイステップの手順を与える。ここで説明する方法は、Jurkat T細胞および活性化ヒトT細胞4-8にCRAC電流を記録するために使用される手続きから導入されました。

Watch this Scientific Journal Video about Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes at JoVE.com

Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

我々は、末梢血単核細胞由来のヒトTリンパ球におけるカルシウム放出、活性化カルシウム（CRAC）電流の全細胞パッチクランプ記録のためのステップバイステップのプロトコルを提供しています。

ヒトTリンパ球におけるカルシウム放出、活性化カルシウム（CRAC）電流のホールセル記録

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated ...

whole-cell-recording-calcium-release-activated-calcium-crac-currents

Research

JoVE Journal

Biology

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

16.0K ビュー.  University of California, Davis. 我々は、末梢血単核細胞由来のヒトTリンパ球におけるカルシウム放出、活性化カルシウム（CRAC）電流の全細胞パッチクランプ記録のためのステップバイステップのプロトコルを提供しています。

ビデオ: Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

カルシウムインジケータ付きのロードショウジョウバエの神経終末

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

生物学

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

レイヤーin vivoでの2光子カルシウムイメージングマウスの2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

光の全細胞録音は網膜スライスにおける興奮性シナプス電流を誘発

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

アストロサイトの細胞膜とグローバル細胞内カルシウム動態の近くの測定

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

遺伝的にコードされたカルシウムのセンサを用いたアポトーシス時のミトコンドリア内カルシウム濃度の動的変更の監視

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

心筋細胞における高速カルシウムフラックスの測定

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

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 ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

フローサイトメトリーによる腎上皮細胞における細胞質ゾルカルシウム測定

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

リアルタイム体内シロイヌナズナカルシウム信号の間に記録して蛍光バイオ センサーを用いた昆虫の餌

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

マウス膵島細胞の亜集団におけるカルシウムダイナミクスのイメージング

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...