This study was supported by the Swiss National Science Foundation (Grant 32003B_127619).

大腿四頭筋生検は、ミトコンドリアを分画遠心分離によって分離されて、そこから麻酔したブタから採取されます。豚は別の実験のために後で使用されます。研究は、実験動物の管理と使用のためにとベルン、スイスの動物ケア委員会の承認を得て国立衛生研究所のガイドラインに従って行われます。 1.骨格筋の均質化とミトコンドリアの単離<ol><li> 消費税麻酔したブタ5〜10グラムの大腿四頭筋標本。本発明のアッセイでは、ブタの骨格筋を使用しています。このプロトコルは、他の種からミトコンドリアを単離することができる（ 例えば 、ラット、マウス、ヒト、 など ）。 注：この手順は、手術を専門とする医療専門家によって行われます。 <ol><li>筋肉内ケタミン（20 mgの/ kg）およびキシラジン（2 mgの/ kg）で鎮静ブタ、麻酔内で誘導される前静脈ミダゾラム（0.5ミリグラム/キログラム、プラスアトロピン0.02ミリグラム/キログラム）。手術中にプロポフォールの連続静脈内注入（1時間当たり4-8ミリグラム/キログラム）で麻酔し、フェンタニル（1時間当たり30μgの/キログラム）を維持。 </li></ol></li><li>直ちに氷冷ミトコンドリアの単離緩衝液（ 表1）の20 mLを含むビーカーに組織試料を浸します。 注：ホモジナイズし、遠心分離中に使用される緩衝液は、冷たい氷であると細胞質ゾルと互換性のイオン性及び浸透強度と生理的に適切なpHを有していなければなりません。 </li><li>分析風袋を計ったバランスのビーカー内の組織サンプルを秤量します。風袋分離バッファの20ミリリットルを入れたビーカーとのバランス。 </li><li>アイスバケットにビーカーを置きます。 注：アイスバケット温度で均質化の手順については、次の手順をすべて実行し、アイスバケットにすべてのバッファを保持します。 </li><li>ビーカーの中に骨格筋をみじん切り罰金scissを使用して3-4分間1〜2ミリメートル小片に（氷上で保持）ORS。 </li><li>氷冷分離バッファ（各20mL洗浄工程）で2回ビーカー内のみじん切り組織をすすいでください。 </li><li> 5 mgのプロテアーゼ/ g組織を含む組織重量（g）当たりの単離緩衝液の10体積の組織を一時停止します。 </li><li>マグネチックスターラー上で氷浴中でビーカーを置き、10分間攪拌します。 </li><li> 0.2％（重量/体積）の脱脂ウシ血清アルブミン（BSA）を補充した組織の重量（g）当り分離緩衝液の追加の10容量のビーカー中の懸濁液を希釈します。 </li><li> 0.2％BSAを補充した単離緩衝液のすべてをデカントし、0.2％BSA（20 mLを各洗浄工程）を補充した氷冷分離緩衝液で2回ビーカー中で組織をすすぎます。 </li><li>脱脂BSAと0.2％を補充した組織の重量（g）当り分離緩衝液の10体積の組織を再懸濁します。 </li><li>ゆったりと乳棒（10ストローク）で（氷上で保持）半自動ガラスホモジナイザーで組織をホモジナイズします。 注：Homogeniz同様に、常に電子組織（ストロークの同じ数、 例えば 、10ストローク）。ストローク数が多いほど、ミトコンドリアを損傷し、切り離すことができます。好ましくは、自動化されたホモジナイザーを用いて組織をホモジナイズします。手動で（および主観）ガラスホモジナイザーでホモジナイズした組織試料は、単離されたミトコンドリアの異なる品質を生成することができます。 </li><li>遠心分離機万×gで10分間4℃でホモジナイズした組織。 注：すべての遠心操作は固定角ローターと遠心機で実行されています。 </li><li> （10ミリリットル/ g組織）血清学的ピペットを用いて上清を捨て、BSAを補充した氷冷分離培地にペレットを再懸濁します。 </li><li>遠心分離機350×gで10分間4℃でサスペンション。 </li><li>血清学的ピペットを用いて上清を予約し、ペレット（細胞破片を）捨てます。 </li><li>細胞debrを除去するために、ガーゼの2層（17スレッド/ cm 2）を介して（氷上で保持）ビーカーに上清をフィルターです。 </li><li>遠心分離機7000×gで10分間4℃で懸濁液を濾過。 </li><li>上清を捨て、7000×gで10分間4℃で0.2％（重量/体積）BSAおよび遠心サスペンションを補充した単離緩衝液（5ミリリットル/ g組織）中の粗ミトコンドリアペレットを再懸濁。 </li><li>上清を捨て、洗浄緩衝液（表1）中の粗ミトコンドリアペレットを再懸濁。遠心分離機7000×gで10分間4℃で再びサスペンション。 </li><li>上清を捨て、7000 X gで10分間4℃で再び洗浄緩衝液および遠心分離中の懸濁液を、粗ミトコンドリアペレットを再懸濁。 </li><li>上清を捨て、洗浄緩衝液1.0mLの中の粗ミトコンドリアペレットを再懸濁。 </li><li>ミトコンドリア懸濁液のタンパク質濃度を決定し、氷上で濃厚ミトコンドリア懸濁液を保ちます。 注記：タンパク質濃度は、任意の標準的な方法を用いて測定することができます。 </li><li>直前アッセイ呼吸計測するために、0.4 mg / mlでミトコンドリアタンパク質の最終濃度に呼吸緩衝液中のミトコンドリア懸濁液を再懸濁します。 </li></ol> 2.高解像度の呼吸計測<ol><li>ピペット高解像度oxygraph室に呼吸緩衝液（ 表1）16の2.1 mL及び連続の安定した酸素フラックス信号まで1時間、37℃でチャンバ内に存在する磁気撹拌棒（700 rpm）を用いて緩衝液をかき混ぜますポーラログラフ酸素センサが得られます。 注：各oxygraph室内のポーラログラフ酸素電極は、酸素濃度を測定し、各チャンバー内の酸素消費量（フラックス）を計算します。酸素濃度と酸素消費速度（フラックス）は、データ収集および分析のためのソフトウェアを使用してコンピュータにオンラインでリアルタイムに表示されます。また、正確で信頼性のある結果を確保するために、楽器の酸素のバックグラウンド補正があるべきです製造業者の指示に従って行いました。 注：呼吸バッファの正確2.1 mLの栓を閉じた後、2.0 mLの最終チャンバ容積の較正のためのチャンバーに添加されます。 </li><li>製造業者のプロトコル17に従ってポーラログラフ酸素センサのエアキャリブレーションを実行します。 注意：空気校正の間、気相に存在して、メディアの酸素濃度は、15〜20分程度で平衡化します。温度、気圧、およびメディア - 酸素濃度の溶解度に応じて算出することができます。 </li><li>エアキャリブレーション後、oxygraph室から呼吸培地を吸引し、oxygraphのチャンバーにステップ1.24から単離されたミトコンドリア懸濁液（0.4 mg / mlでミトコンドリアタンパク質）の2.1 mLを加え。 注：呼吸バッファの容量が設定し、機器とメーカーによって異なります。高解像度の呼吸計測のために、呼吸府の2.1 mLのfferはストッパーを閉じた後、2.0 mLの最終チャンバ容積の較正のためのチャンバーに添加されます。 </li><li>ストッパーの挿入によってoxygraph室を閉じます。 </li><li>連続して37℃のチャンバ内に存在する磁気攪拌棒（700 rpm）を用いて、ミトコンドリア懸濁液を撹拌し、安定した酸素フラックス信号が達成されるまで、3〜5分間、ベースライン時に細胞呼吸を記録。 注：酸素濃度と酸素フラックス信号はデータ収集および分析17のためのソフトウェアを使用してコンピュータにオンラインでリアルタイムに表示されます。 </li><li>その後、以下の標準的なプロトコルを使用して、ストッパのチタン注入ポートを介してミトコンドリア呼吸の基質と阻害剤を注入します。 </li></ol> 3.複合体I-依存呼吸<ol><li>ステップ2.1から2.5に記載の手順に従って、単離されたミトコンドリア懸濁液（0.4ミリグラム/ ml）を含むoxygraph室を準備し、安定した信号を待ちます。 </li><li>チャンバーストッパーのチタン注入口から分離されたミトコンドリア懸濁液（0.4ミリグラム/ ml）を含むoxygraph室に0.8 Mリンゴ酸（5mMの最終濃度）の12.5μLと2 Mグルタミン酸（10mMの最終濃度）の10μLを注入し、安定した酸素フラックス信号が達成されるまで、3〜5分間、細胞呼吸を記録。 注：以下の手順のすべての注射は注射器を使用してストッパーのチタン注入ポートを介して実行されます。 </li><li>チャンバーストッパーのチタン注入口からoxygraph室に0.05 M ADP（0.25ミリモル）の10μLを注入し、酸素フラックス信号が増加するまで、ミトコンドリア呼吸を記録し、その後減少し、安定化（3-5分）。 注：ADP添加後の呼吸の高原は、ADP濃度が高く、飽和したことを示しています。呼吸中に使用されるミトコンドリアの量に応じて、ADP濃度を滴定されるべきです安定した状態3の酸素消費量（最大呼吸）のために。ミトコンドリア懸濁液にADPの添加は、（状態3呼吸）を増加させる酸素消費量の増加及び酸素フラックスシグナルを誘発します。 ADPが消費された後、酸素フラックス信号は（状態4呼吸）が低くなります。 </li></ol> 4.複合体II依存の呼吸<ol><li>ステップ2.1から2.5に記載されており、安定した信号を待つ手順に従って、単離されたミトコンドリア懸濁液（0.4ミリグラム/ ml）を含むoxygraph室を準備します。 </li><li>注射器を用いてチャンバストッパーのチタン注入ポートを介してoxygraph室に0.2 mMのロテノン（0.2μM）「注意」の2μLを注入し、安定した酸素フラックス信号が達成されるまで、3〜5分間、細胞呼吸を記録。 注：ロテノンは危険毒であり、ヒトの健康に重大な影響を有することができます。 </li><li> oxygraphのCHに1 Mコハク酸（10mMの）の20μLを注入アンバーと安定した酸素フラックス信号が達成されるまで、3〜5分間、ミトコンドリア呼吸を記録します。 </li><li>チャンバーストッパーのチタン注入口からoxygraph室に0.05 M ADP（0.25ミリモル）の10μLを注入し、酸素フラックス信号が増加するまで、細胞呼吸を記録し、安定化、その後減少し、安定化（3-5分）。 注：ミトコンドリア懸濁液にADPの添加は、（状態3呼吸）を増加させる酸素消費量の増加及び酸素フラックスシグナルを誘発します。 ADPが消費された後、酸素フラックス信号は（状態4呼吸）が低くなります。 </li></ol> 5.複合体IV依存の呼吸<ol><li>手順プロトコルの2.5から2.1と安定した信号を待つに記載の手順に従って、単離されたミトコンドリア懸濁液を含むoxygraph室（0.4ミリグラム/ mL）を準備します。 </li><li>その後、0.8 mMのアスコルビン酸（1ミリモル）の2.5μLを注入。その直後に0.2の2.5μLを注入M TMPD（0.25ミリモル）および0.05 M ADP（0.25ミリモル）の10μLoxygraph室へと酸素フラックス信号が増加するまで、細胞呼吸を記録し、（3-5分）を安定化させます。 </li><li>最後にoxygraph室に1 Mアジ化ナトリウム（5 mM）の「警告」の10μLを注入し、酸素フラックス信号が減少するまで、細胞呼吸を記録し、安定化させます。 注：アジ化ナトリウムは危険毒であり、ヒトの健康に重大な影響を有することができます。 </li></ol> 6.チトクロームCテスト<ol><li>手順プロトコルの2.5から2.1と安定した信号を待つに記載の手順に従って、単離されたミトコンドリア懸濁液を含むoxygraph室（0.4ミリグラム/ mL）を準備します。 </li><li>チャンバーストッパーのチタン注入口から分離されたミトコンドリア懸濁液（0.4ミリグラム/ ml）を含むoxygraph室に0.8 Mリンゴ酸（5mMの最終濃度）の12.5μLと2 Mグルタミン酸（10mMの最終濃度）の10μLを注入し安定した酸素フラックス信号が達成されるまで、3〜5分間、細胞呼吸を記録。 </li><li>チャンバーストッパーのチタン注入口からoxygraph室に0.5 M ADP（2.5 mM）の10μLのを注入し、酸素フラックス信号が増加するまで、ミトコンドリア呼吸を記録し、その後減少し、安定化（3-5分）。 </li><li>最後に、oxygraph室に4mMのシトクロムc（10μM）の5μLを注入し、安定した酸素フラックス信号が達成されるまで5分間細胞呼吸を記録。 </li></ol>

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Biol. 810, 25-58 (2012).</li><li>Picard, M., 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=Mitochondrial+structure+and+function+are+disrupted+by+standard+isolation+methods.">Mitochondrial structure and function are disrupted by standard isolation methods.</a> PLoS One. 6 (3), e18317 (2011).</li><li>Picard, M., 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=Mitochondrial+functional+impairment+with+aging+is+exaggerated+in+isolated+mitochondria+compared+to+permeabilized+myofibers.">Mitochondrial functional impairment with aging is exaggerated in isolated mitochondria compared to permeabilized myofibers.</a> Aging Cell. 9 (6), 1032-1046 (2010).</li><li>Graham, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+a+crude+mitochondrial+fraction+by+density-gradient+centrifugation.">Purification of a crude mitochondrial fraction by density-gradient centrifugation.</a> Curr. Protoc. Cell. Biol. Chapter 3. 3, (2001).</li><li>Franko, A., 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=Efficient+isolation+of+pure+and+functional+mitochondria+from+mouse+tissues+using+automated+tissue+disruption+and+enrichment+with+anti-TOM22+magnetic+beads.">Efficient isolation of pure and functional mitochondria from mouse tissues using automated tissue disruption and enrichment with anti-TOM22 magnetic beads.</a> PLoS One. 8 (12), 382392 (2013).</li><li>Pecinov&#225;, A., Drahota, Z., N&#367;skov&#225;, H., Pecina, P., Hou&#353;t&#283;k, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+basic+mitochondrial+functions+using+rat+tissue+homogenates.">Evaluation of basic mitochondrial functions using rat tissue homogenates.</a> Mitochondrion. 11 (5), 722-728 (2011).</li><li>Lombardi, A., 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=Characterisation+of+oxidative+phosphorylation+in+skeletal+muscle+mitochondria+subpopulations+in+pig:+a+study+using+top-down+elasticity+analysis.">Characterisation of oxidative phosphorylation in skeletal muscle mitochondria subpopulations in pig: a study using top-down elasticity analysis.</a> FEBS Lett. 475 (2), 84-88 (2000).</li></ol>

The authors have nothing to disclose.

本研究において、我々は、無傷でしっかりこのような高解像度の呼吸計測等の機能的研究のために使用することができる差動遠心分離によって骨格筋のミトコンドリアを結合、高品質を単離するためのプロトコルを説明します。 無傷と密結合ミトコンドリアを単離するために、本プロトコル内で考慮すべきいくつかの重要なポイントがあります。骨格組織を採取した後?...

ミトコンドリア生体エネルギーと呼吸容量は透過性細胞または繊維ではなく、また、単離されたミトコンドリアだけでなく研究することができます。本研究では、高解像度の呼吸計測測定のために分画遠心法を用いて、無傷の骨格筋のミトコンドリアを単離するためのプロトコルを説明します。 呼吸計測のための完全なミトコンドリアを単離するために、組織が均質化され、ミトコンドリアは、従来の分画遠心法により単離されます。遠心分離法は、組織ホモジネートの連続遠心分離（増速の直列）に基づいて差は最初、ほぼ70年前に1 Palladeおよび共同研究者によって導入されました。組織は、最初にハサミを使って細かく刻み、ゆったり乳棒でガラスホモジナイザーで機械的に均質化されます。その後、ホモジネートを低速と切れ目のない組織が含まれている得られたペレットを、携帯で遠心分離され、破片および核は破棄されます。その後、上清を高速で数回遠心分離し、ミトコンドリア富化画分が回収されます。ミトコンドリアを単離するための差動遠心分離法の利点は、ということです：ⅰ）メソッドは高速であり、ミトコンドリアは（呼吸実験は可能な限り迅速として行われるべきである）1〜1.5時間以内に単離することができます。 ⅱ）それは安価です。そしてiii）それは非常に効率的であり、微分遠心分離によって得られたミトコンドリアは、呼吸計測分析のために十分に純粋です。ミトコンドリアを単離するための差動遠心分離法の欠点は、ⅰ）ミトコンドリアが均質化中に損傷し、非結合を受ける可能性があることです。 ⅱ）他の細胞成分とミトコンドリアの汚染は（）、さらに追加の遠心分離工程でミトコンドリアのペレットを洗浄することにより解決することができます。 ⅲ）差動遠心分離工程の間に、異なるミトコンドリア亜集団、 例えばを選択する可能性を、MIT下の密なとochondriaは7除外することができます。およびiv）ミトコンドリアの細胞周囲の不足しているとだけ理論的な最大の呼吸を測定することができます。呼吸計測分析のためにミトコンドリアを単離するための別の方法は、密度勾配遠心分離2です。この技術では、組織抽出物（遠心管の底部でのより高い密度を有する）スクロース溶液またはPercoll勾配の上に積層され、一定の速度で遠心分離し、ミトコンドリアが、それらに応じて、他の細胞成分から分離することを引き起こします密度。この方法は、多くの場合、シナプトソームから非常に低い汚染に脳ミトコンドリアを単離するために使用されます。しかしながら、密度勾配遠心分離によって単離されたラット肝ミトコンドリアは非常に他の細胞小器官3で汚染されています。この方法の制限の一つは、遠心分離管に存在するショ糖勾配がそう破裂かもしれないということです私ミトコンドリア（浸透圧ショック）。 組織の種類に応じ。差動遠心分離による無傷ミトコンドリアの単離のために考慮すべきいくつかの重要な要因があります。最初の必要性は穏やかな方法で組織を均質化することです。例えば、腎臓、脳や肝臓などの軟組織は、均質化の間に適用される穏やかな機械的な力を必要とします。これは、はるかに強い機械的な力を必要とし、心臓および骨格筋などの硬組織とは対照的です。細分化した組織は、通常、組織を柔らかくするために均質化前にプロテアーゼで処理されます。均質化および遠心分離の間に使用されるすべての緩衝液は、冷たい氷であると細胞質ゾル4、5と互換性のイオン性及び浸透強度と生理的に適切なpHを有していなければなりません。 分離されたミトコンドリアの生体エネルギーを研究する利点の一つは、細胞形質膜はpermeabiである必要はないということですこのようなミトコンドリア外膜の完全性を損なう可能性がありますジギトニンまたはサポニン4、6、などの界面活性剤とlized。単離ミトコンドリアの別の利点は、酸素消費量としてミトコンドリアの機能の分析を妨害し得る他のサイトゾル因子の欠如です。単離されたミトコンドリアを用いての欠点は、単離されたミトコンドリア7,8の良好な収率を得るために遠心分離工程中のあるミトコンドリアの集団の可能な選択、均質化の間にミトコンドリアの損傷、および生物学的サンプルの大量の要求です。 分離手順の後、ミトコンドリア複合体I、II-IV及び依存（状態2,3および4）の呼吸速度は、高解像度の呼吸計測を使用して決定されます。複合体I-駆動呼吸のために、グルタミン酸リンゴ酸は、アデノシン二リン酸（ADP）を加え、続いています。複合体II駆動型の呼吸のために、コハク酸は、ADPを添加し、続いています。複雑なIV-駆動呼吸については、アスコルビン酸及びtetramethylphenylendiamine（TMPD）はADP 9、10、11、12、続いて添加されます。状態2は、単独で、基質の存在下での酸素消費量を指します。状態3は、基板と、ADPの存在下での酸素消費量を指します。状態4は、ADPの枯渇後の酸素消費量を指します。呼吸調節比（RCR）は、酸素消費量のATP生産の結合の指標であり、状態3及び状態4 13、15との間の比として計算されます。 要約すると、我々は、差動遠心分離により、機能と無傷の骨格筋のミトコンドリアを単離し、これらの単離されたmitochonを使用するプロトコルを記述しますこのような高解像度の呼吸計測などの機能や生体エネルギー研究のためdria。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ADP</td>
			<td>Sigma</td>
			<td>A 4386</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma</td>
			<td>A 8674</td>
			<td>Chemical, dissolve in ethanol</td>
		</tr>
		<tr>
			<td>Ascorbate</td>
			<td>Merck</td>
			<td>1.00127</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma</td>
			<td>A 7699</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A 6003</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>fluka</td>
			<td>3779</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma,</td>
			<td>G 1626</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma</td>
			<td>H 7523</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Merck</td>
			<td>1.04936</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Merck</td>
			<td>1.04873</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>K-lactobionate</td>
			<td>Sigma</td>
			<td>L 2398</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M 9272</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Morpholinopropane sulphonic acid (MOPS)</td>
			<td>Merck</td>
			<td>1.06129</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>O2k-Core: Oxygraph-2k&#160;</td>
			<td>Oroboros Instruments</td>
			<td>10000-02</td>
			<td>High-resolution respirometry instrument</td>
		</tr>
		<tr>
			<td>Proteinase, bacterial</td>
			<td>Sigma</td>
			<td>P 8038</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma</td>
			<td>R 8875</td>
			<td>Chemical, dissolve in ethanol</td>
		</tr>
		<tr>
			<td>Succinate</td>
			<td>Sigma</td>
			<td>S 2378</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Schuett homogen-plus semiautomatic homogeniser&#160;</td>
			<td>schuett-biotec GmbH</td>
			<td>3.201 011</td>
			<td>Tissue homogenizer</td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma</td>
			<td>T 8691</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>TMPD</td>
			<td>Sigma</td>
			<td>T 3134</td>
			<td>Chemical</td>
		</tr>
	</tbody>
</table>

isolation of intact mitochondria from skeletal muscle by differential centrifugation for high-resolution respirometry measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

複合体I-依存呼吸 単離されたミトコンドリア複合体I依存呼吸数（状態2、図3、図4）は、高解像度の呼吸計測（ 図1、代表的な図）を用いて決定されます。ミトコンドリア複合体I基板、グルタミン酸とリンゴ酸は、ADPを加え、続いています。状態2は、単独で、基質の存在下での酸素消費量を指し?...

Watch this Scientific Journal Video about Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements at JoVE.com

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

高解像度の呼吸計測測定のための差動遠心分離することにより骨格筋から無傷ミトコンドリアの単離

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

isolation-intact-mitochondria-from-skeletal-muscle-differential

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

18.0K ビュー.  Inselspital, Bern University Hospital. Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

ビデオ: Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

骨格筋からのミトコンドリア単離

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

生物学

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

骨格筋からの前駆体の精製

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

成人骨格筋からの個々の筋線維とその衛星細胞の単離および培養

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

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 ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

ヒト骨格筋からの血管由来の多能前駆体の単離

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

エピジェネティックリプログラミングによるヒトES細胞からMyospheresの生成

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

組織解離と差動濾過により移植のためにミトコンドリアの迅速な単離および精製

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

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

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

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

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

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

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

透過処理と無傷の細胞でミトコンドリア機能を評価するために高解像度呼吸計測

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

天然化合物の存在下でそのまま β 細胞のミトコンドリア機能を測定するための高解像度の呼吸

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...