The Fralin Life Science Research Institute and The Metabolic Phenotyping Core at Virginia Tech supported this work.

動物実験は、動物実験によって承認されたプロトコルの下で行われ、バージニア工科大学で委員会を使用しました。 1.セットアップ（時間：〜45分） <ol><li> 37℃の水浴中で0.25％トリプシン、単離ミトコンドリアのバッファ（IBM）1 IBM2の凍結貯蔵を解凍。 </li><li>ガラス製品をすすぎ、高純度水に続いて70％エタノール中で楽器を解剖。 </li><li> 4部IBM1で1部のトリプシンを希釈することにより、0.25％のトリプシンの株式0.05％トリプシン溶液を準備します。 </li><li>スクリューキャップ1.5 mlのマイクロ遠心チューブ内：（100比1）細胞溶解バッファーでプロテアーゼおよびホスファターゼ阻害剤カクテルを混ぜます。 </li><li>アリコートを5 mlの15 mlのプラスチックチューブに、0.05％トリプシン（5ミリリットル/マウス）。 </li><li> 80 RPMにモータ駆動の組織ホモジナイザーを設定します。 </li></ol>骨格筋ミトコンドリアの2分離（時間：〜90分） <ol><li> CO 2でマウスを安楽死させます</su頸椎脱臼に続いてB&gt;吸入、。 </li><li>大腿四頭筋と腓腹筋から赤筋を削除するには、以下の手順で説明するようにヒラメ（〜75-100 mgの合計）が含まれています： <ol><li>マウスの方の皮膚の皮をむきます。 </li><li>大腿四頭筋原点の上に脂肪パッドを取り外します。細かい傾いてハサミで膝蓋骨に接続されている四頭筋腱をカットします。 注意：大腿四頭筋は、大腿骨近位部の前方部分に、その解剖学的位置によって識別されます。 </li><li>骨から大腿四頭筋を解放するために、大腿動脈を回避しながらゆっくりと、骨と大腿四頭筋腱膜の間を切り取ります。大腿四頭筋を解放し、冷PBSで大腿四頭筋を配置するために、大腿骨上の原点で細かい傾いてハサミで腱をカットします。 </li><li>ハサミで大腿四頭の上に目に見える脂肪組織を削除します。大腿骨の上に位置された筋肉の部分はUを向くように上で大腿四頭筋をフリップP。ファニングの運動でピンセットで大腿四頭筋を開きます。チルドIBM1の5ミリリットルを含むビーカー中に微細な先端ハサミと場所で各葉から2可視赤色筋肉部分を削除します。 注意：大腿四頭筋は、各葉の側縁付近に位置する赤筋のストリップで2つのローブとして表示されます。 </li><li>細かい先端に付いたハサミでアキレス腱を覆う皮膚を切り、マウスに向かって皮をはがし。露出したアキレス腱を切って、マウスの体内に向けて筋肉を剥離。 </li><li>腓腹筋を解放し、冷却PBS中の腱に取り付けた腓腹筋を配置するために、微細な先端ハサミで大腿骨の外側顆および内側顆で腱をカットします。 </li><li>以上の腓腹筋を裏返し、ヒラメ筋を剥離し、冷却IBM1の5ミリリットルを入れたビーカーに入れます。ファンは腓腹筋を開きます。細かい傾いてはさみで3種類の視覚的な赤い筋肉部（二つの側方および1内側表面的な赤のストリップ）を削除しますSとは、冷却IBM1の5ミリリットルを含むビーカーに転送します。 </li></ol></li><li>マウスの他の脚部から赤筋の別〜75-100 mgの削除（ステップ2.2）およびプロテアーゼおよびホスファターゼ阻害剤カクテルおよび細胞溶解緩衝液を用いて1.5 mlのマイクロ遠心チューブに入れる（50mMのTris-HCL、1 mMのEDTA、150mMのNaCl、1％ドデシル硫酸ナトリウム、0.5％デオキシコール酸ナトリウム、1％ポリオキシエチレン（9）ノニルフェニルエーテルは、直ちにウェスタンブロッティングにおける後の使用のために液体窒素中でこの第二のサンプルをフラッシュ凍結分岐（ステップ6.1参照）。 </li><li>大きなバケツに氷上で予冷し、平らなプラスチック表面を置きます。プラスチック表面上にIBM1の低下を注ぎ、IBM1滴に区分赤筋（ステップ2.2.4および2.2.7）のすべてを配置するためにピンセットを使用しています。カミソリごとに40秒を変更することにより、3つのシングルエッジかみそりの刃を使用して、2分間の筋肉組織をミンチ。 </li><li>目の上にプラスチック表面を保持することによって、新鮮なIBM1 5mlで新しいビーカーに刻んだ組織を転送Eビーカーやかみそりの刃でビーカーに筋肉をこします。このソリューションを取り、50mlコニカルチューブ上に配置さ100μmのセルストレーナーを介して排出します。 </li><li>繊細な作業ワイパーで組織をブロットした後、0.05％トリプシン溶液5mlにそれを転送します。タスクワイパーから組織を除去するためにピンセットを使用してください。 </li><li> 0.05％トリプシン溶液中で30分間氷上で筋肉組織をインキュベートします。 </li><li> 4℃で3分間200×gで15ミリリットルコニカルチューブ含むトリプシン/筋肉の混合物をスピン。 </li><li>廃棄物容器にトリプシン上清を注ぎ、氷冷IBM1 3mlでペレットを再懸濁。 45ミリリットルのガラスホモジナイザーチューブに組織を移します。残りのサンプルを収集し、ガラスホモジナイザーチューブにこれを追加するために、別の1.5ミリリットルIBM1で15ミリリットルにコニカルチューブを洗浄します。 </li><li>ガラスホモジナイザーをビーカー内で最​​小限に移動して、氷を入れたビーカーやプラスチック容器満たし途中にガラスホモジナイザーチューブを置きます。 </li><li&gt; 80 rpmで10パスしてモータ駆動の組織ホモジナイザーに接続されているPTFE製乳棒で組織/ IMB1混合物を均質化します。 〜2秒間、各パスの底を持ってください。 </li><li>新しい15ミリリットルコニカルチューブに組織ホモジネートを移し、IBM1 6.5 mlのガラスホモジナイザーチューブをすすいでください。組織ホモジネートを含む15ミリリットルコニカルチューブにIBM1を注ぎます。 </li><li> 4℃で10分間700グラムで15ミリリットルコニカルチューブをスピン。静かにガラスの高強度遠心管に上清を注ぐし、ペレットを廃棄します。 </li><li> 4℃で10分間8000×gで、上記のステップからの上清をスピン。 </li><li> IBM1の上清を除去し、ゆっくりIBM2500μlのを追加することにより、ペレットを再懸濁。ピペットチップの先端をカットし、軽く運動を混合し、攪拌しながらIBM2でペレットを均質化します。ペレットが完全に中断された後、混合物にIBM2の別の4.5ミリリットルを追加します。 注：tと大きなペレット片を破壊しながら、過度の摩砕を避けます彼は、ミトコンドリア膜を損傷することがあります。 </li><li> 4℃で10分間8000×gでIBM2 /組織ホモジネートをスピン。 注：残留BSAは、総タンパク質含有量に寄与することができるので、このステップは、ステップ4において、単離されたミトコンドリアのより正確なタンパク質定量のためのクリーンな高強度遠心管中で繰り返してもよいです。 </li><li>上清を除去し、静かに、しかし完全に遮断、その点にピペットチップでIBM2の225μlの増分を追加することにより、ペレットを再懸濁。ゆっくりIBM225μlのそれぞれの添加後にペレットを攪拌、混合します。氷の上でこのミトコンドリアの在庫を置きます。 </li></ol>全組織溶解物の3均質化（時間：〜45分は、ステップ7の間に行うことができます） <ol><li>それがステップ2.3から液体窒素の中に入れたから筋肉を外し、完全に解凍するまで室温でインキュベートします。 </li><li>細かい傾いてハサミで氷上で〜30秒のための組織をみじん切り。 </li><li>ジルコ二100μlのスクープを追加みじん切りの組織が含まれているステップ3.2からのスクリューキャップ1.5 mlのマイクロ遠心チューブにニウム酸化物ビーズ。 4-6（メディア設定）の速度設定で2 5 3分のサイクルを使用して、ビーズミル組織ホモジナイザー中で組織をホモジナイズします。 </li><li> 4℃で10分間12,000×gでステップ3.3からの混合物をスピン。スピンした後、千μlのピペットチップを使用して上清を除去し、1.5 mlのマイクロ遠心チューブに移します。ペレットを捨て、氷の上に上清を配置します。 </li></ol> 4.タンパク質の決意（時間：〜30分） <ol><li>製造業者の仕様に従って、BCAタンパク質アッセイキットを使用して、全組織溶解液（ステップ3.4）とミトコンドリアの株式（ステップ2.17）のタンパク質濃度を決定します。 </li></ol> 5.ミトコンドリア膜の完全性、クエン酸シンターゼ（CS）活動（時間：〜15分） <ol><li>高純度水におけるミトコンドリアの株式の20希釈液：二つの別々の1を作ります。追加一つのサンプルにトリトン100-Xの0.1％と低く設定（765ワット、2の振幅）でそれを超音波処理します。氷の上の他の希釈のままにしておきます。超音波処理した後、氷にサンプルを返します。 </li><li>でクエン酸シンターゼアッセイを行う両方の非超音波処理し、以前に記載されているように11,12を分光光度アッセイを用いてミトコンドリアの希釈液を超音波処理しました。 </li><li>以下の式を用いて、無傷のミトコンドリア膜の割合を決定します。 （超音波処理CS活性÷非超音波処理CS活性）* 100 </li></ol> 100- N（上から達成パーセント） 6.ミトコンドリアの分離品質。ウェスタンブロッティング（時間：ラボプロトコルによります） <ol><li>以前に説明したように12全組織溶解物および単離ミトコンドリアにウェスタンブロッティングを行います。 注意：GAPDHのための使用の一次抗体を（1：1,000希釈5％のBSA / TBS-T）とCOXIV（1：1,000希釈の5％無脂肪ミルク/ TBS-T）（1それぞれ、抗ヤギ、ウサギ二次抗体が続く：10,000）。 </li></ol> 7.呼吸アッセイを実行します（時間：〜90分） <ol><li>前述したように、マイクロプレートベースのO 2消費の測定技術を用いて所望の呼吸アッセイを実行します（関連記事やロジャースを参照ら8）。 </li><li>ステート40（RCR）によって状態3を分割または州40によって状態の3Uを分割することにより、呼吸制御比（RCR）を決定します。 RCRを決定する際に、ポイントツーポイントトレースからミドル（平均）点、または最高の（状態3）および最低宿泊料金（状態40）のいずれかを使用してください。 </li></ol>

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George Rogers is an employee of Seahorse Bioscience that produces the instrument for which this protocol was modified.

本明細書に提示する方法は、マウス骨格筋の最小量（〜75〜100ミリグラム）から、ミトコンドリアの単離手順の詳細な説明を提供します。この単離手順は、O 2消費率、RCR値、最大のクエン酸シンターゼ活性およびイムノブロットからのタンパク質の発現によって証明されるように、高機能、純粋なミトコンドリア（〜450μgの）を生成することができます。重要なことには、この手順か...

The primary function of mitochondria is to produce ATP from oxidative phosphorylation. However, mitochondria have many other important cellular functions including but not limited to: the production and detoxification of reactive oxygen species, the regulation of cytoplasmic and mitochondrial calcium, organelle trafficking, ionic homeostasis, and involvement in apoptosis1,2. Therefore, it is not surprising that dysfunctional mitochondria play a role in many disease pathologies, such as aging, neurodegenerative diseases, cardiovascular disease, cancer, obesity, and diabetes3,4. Importantly, skeletal muscle mitochondria specifically are involved in many of these pathologies3-5.
Mitochondrial respiration assays using isolated mitochondria allow for the assessment of electron transport chain and oxidative phosphorylation function, and the determination of mechanism(s) of action of drugs and proteins that modulate metabolism. Mitochondrial isolation procedures exist for multiple tissue and cell types for a variety of species6,7. However, these procedures often require large quantities of tissue/cells for a high quality mitochondria yield necessary for classic respirometric assays. 
Microplate based respirometirc assays allow for high throughput measurements using minimal quantities of isolated mitochondria, often just several &#181;g per well8. Therefore, we present a modification of previously published methods7 to allow for high quality mitochondria to be isolated from smaller quantities of mouse skeletal muscle for use in microplate based respirometirc assays. In addition, methods are provided to establish the quality of the mitochondrial isolation preparation and the integrity of the mitochondrial membranes. Given that skeletal muscle mitochondria are involved in many pathological conditions, the measurement of O2 consumption in mechanistically driven studies is becoming more prevalent in biomedical research9,10.

<table><tbody><tr><td>Essentially Fatty</td><td>Sigma Aldrich</td><td>A6003</td><td>N/A</td></tr><tr><td>Acid Free- BSA</td><td></td><td></td><td></td></tr><tr><td>Tris/HCl</td><td>Promega</td><td>H5123</td><td>N/A</td></tr><tr><td>KCL</td><td>Sigma Aldrich</td><td>P9541</td><td>N/A</td></tr><tr><td>Tris Base</td><td>Promega</td><td>H5135</td><td>N/A</td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>E6511</td><td>N/A</td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E4378</td><td>N/A</td></tr><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>S7903</td><td>N/A</td></tr><tr><td>D-Mannitol</td><td>Sigma Aldrich</td><td>63559</td><td>N/A</td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Thermo Scientific</td><td>25200-056</td><td>N/A</td></tr><tr><td>Sodium Chloride&lt;br/&gt; White Crystals or Crystalline Powder&lt;br/&gt; &amp;ge;99.0 %</td><td>Fisher Scientific</td><td>BP3581</td><td>N/A</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma Aldrich</td><td>L3771&amp;nbsp;</td><td>N/A</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma Aldrich</td><td>D6750&amp;nbsp;</td><td>N/A</td></tr><tr><td>Polyoxyethylene (12) nonylphenyl ether, branched</td><td>Sigma Aldrich</td><td>238651</td><td>N/A</td></tr><tr><td>Single Edge Razor Blades</td><td>Fisher Scientific</td><td>12-640</td><td>N/A</td></tr><tr><td>Falcon- 100 uM Nylon Cell Strainers</td><td>Fisher Scientific</td><td>352360</td><td>N/A</td></tr><tr><td>Halt Protease &amp;amp; Phosphatse Inhibitor Cocktail</td><td>Thermo Scientific</td><td>1861284</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes with screw cap</td><td>Thermo Scientific</td><td>3474</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Zirconium Oxide beads</td><td>Fisher Scientific</td><td>C9012112</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>GAPDH antibody (1D4)</td><td>Santa Cruz Biotechnology</td><td>sc-59540</td><td>N/A</td></tr><tr><td>Anti- COXIV antibody</td><td>Cell Signaling</td><td>4844s</td><td>Any mitochondrial inner membrane protein will suffice</td></tr><tr><td>Peroxidase conjugated affinipure Donkey, Anti Rabbit IgG (H+L)</td><td>Jackson ImmunoResearh</td><td>711-035-152</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Peroxidase conjugated affinipure Goat, Anti Mouse IgG (H+L)</td><td>Jackson ImmunoResearh</td><td>115-001-003</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Triton-X100</td><td>Sigma Aldrich</td><td>X100</td><td>N/A</td></tr><tr><td>Pierce BCA Protein Assay Kit&amp;nbsp;</td><td>Thermo Scientific</td><td>23225</td><td>N/A</td></tr><tr><td>Pyruvic Acid, 98%</td><td>Sigma Aldrich</td><td>107360</td><td>Store at 4 &amp;deg;C,pH to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td>Succinic Acid</td><td>Sigma Aldrich</td><td>S9512</td><td>Store at room temperature, pH to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td>L(-) Malic Acid, BioXtra, &amp;ge;95%</td><td>Sigma Aldrich</td><td>M6413</td><td>Store at room temperature, to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td>L-Glutamic acid</td><td>Sigma Aldrich</td><td>G1251</td><td>Store at room temperature, to 7.4 with KOH prior to use in respirometric assay, to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Palmitoyl L-carnitine chloride</td><td>Sigma Aldrich</td><td>P1645</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Oligomycin A, &amp;ge; 95% (HPLC)</td><td>Sigma Aldrich</td><td>75351</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Carbonyl cyanide 4-(trifluoromethoxy)</td><td>Sigma Aldrich</td><td>C2920</td><td>Store at 2-8 &amp;deg;C</td></tr><tr><td>phenylhydrazone</td><td></td><td></td><td></td></tr><tr><td>&amp;ge;98% (TLC), powder [FCCP]</td><td></td><td></td><td></td></tr><tr><td>Antimycin A from streptomyces sp.</td><td>Sigma Aldrich</td><td>A8674</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Adenosine 5&amp;prime;-diphosphate monopotassium salt dehydrate [ADP]</td><td>Sigma Aldrich</td><td>A5285</td><td>Store at -20 &amp;deg;C, to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Rotenone</td><td>Sigma Aldrich</td><td>R8875</td><td>Store at room temperature</td></tr></tbody></table>

isolation of mitochondria from minimal quantities of mouse skeletal muscle for high throughput microplate respiratory measurements

機能不全骨格筋ミトコンドリアは、加齢、肥満およびII型糖尿病で観察し、変化した代謝の役割を果たしています。分離されたミトコンドリア調製物からのミトコンドリア呼吸アッセイは、薬物や代謝を調節するタンパク質の作用メカニズム（複数可）のミトコンドリア機能の評価だけでなく、決意を可能にします。現在の単離方法は、多くの場合、呼吸アッセイに必要な高品質のミトコンドリアを得るために組織を大量に必要とします。本明細書に提示される方法は、（〜450μgの）は、高スループットの呼吸測定に使用するために、マウスの骨格筋の最小量（〜75-100ミリグラム）から単離することができる方法を高品質精製ミトコンドリア説明します。私たちは、分離法は、分光光度法、クエン酸合成酵素活性を測定することにより、92.5±2.0％、完全なミトコンドリアをもたらすことを決定しました。また、単離ミトコンドリアにおけるウェスタンブロット分析は、cytosoのかすかな発現をもたらしましたLICタンパク質、GAPDH、およびミトコンドリアタンパク質、COXIVの強い発現。単離されたミトコンドリアにおいて顕著GAPDHのバンドが存在しないことは、単離手順の間の非ミトコンドリア源からの少量の汚染の指標です。最も重要なのは、O 2消費率の測定マイクロプレートベースの技術とし、結合された呼吸アッセイのための呼吸調節比（RCR）を決定するには、高度（RCRを、全てのアッセイのための&gt; 6）に接続ショーや機能ミトコンドリア。結論として、独立したミンチ工程の追加とはかなり以前に報告された方法のモーター駆動均質化速度を低下させることにより、高機能と呼吸性の高い結合されたミトコンドリアになり、マウス骨格筋の少ない量から高品質、精製されたミトコンドリアの単離を可能にしましたマイクロプレートベースrespirometircアッセイ中に。

クエン酸シンターゼはミトコンドリア内膜に位置しており、したがって、無傷の膜を有するミトコンドリアの懸濁物に存在してはならないので、クエン酸合成酵素活性は、膜の完全性のための尺度として役立つ。 図1を超音波処理と比較して、非超音波処理し、ミトコンドリア試料中のクエン酸合成酵素活性を示し、同じ分離からのサンプル。クエン酸シンターゼ活性（P &lt;0.01?...

Watch this Scientific Journal Video about Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements at JoVE.com

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

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 ...

isolation-mitochondria-from-minimal-quantities-mouse-skeletal-muscle

Research

JoVE Journal

Biology

12.7K ビュー.  Virginia Tech. 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.

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

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

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骨格筋の最小量から単離されたミトコンドリアを使用して

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

発生生物学

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.

Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function. Therefore, protein import serves as a barometer of cellular health.

骨格筋ミトコンドリアのタンパク質輸入能の測定

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, ...

生化学

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

経皮針生検によって得られた骨格筋組織から単離されたミトコンドリアの調製と呼吸計測評価

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

医学

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

大人と胚骨格筋のMicroexplantの文化と骨格筋幹細胞の単離

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

生物学

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 ...

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 ...

<meta charset="utf8"></head><body>高品質の生体エネルギー研究のためのげっ歯類からのミトコンドリア単離

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

<meta charset="utf8"></head><body>著者スポットライト:心不全と生体エネルギー学におけるミトコンドリアリン酸カルシウムの役割の解明

Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution respirometry.

げっ歯類の心臓組織からの堅牢なミトコンドリア単離 

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

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 ...

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

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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骨格筋の最小量から単離されたミトコンドリアを使用して

骨格筋ミトコンドリアのタンパク質輸入能の測定

経皮針生検によって得られた骨格筋組織から単離されたミトコンドリアの調製と呼吸計測評価

大人と胚骨格筋のMicroexplantの文化と骨格筋幹細胞の単離

骨格筋からのミトコンドリア単離

骨格筋からの前駆体の精製

げっ歯類の心臓組織からの堅牢なミトコンドリア単離

げっ歯類の心臓組織からの堅牢なミトコンドリア単離

げっ歯類の心臓組織からの堅牢なミトコンドリア単離

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