この作品は、国立眼研究所（R01 EY12998）からFH Andradeさんへの助成金によって支えられている。

1。バッファーの調製<ol><li>遠心5804Rの電源を入れ、4℃に設定37℃にIsotemp 3006D水浴とセットの電源を入れ</li><li>筋肉の分離の前に以下のソリューションを準備します。 <ul><li> PBS：蒸留水（5粒/リットル）にリン酸緩衝生理食塩水（PBS）タブレットを溶かす。よく混ぜる。 </li><li> PBSを加えた10mMのEDTA：100mlの溶液を調製するためには、PBSの98 mlに500 mMのEDTAの2 mlを加える。 </li><li> 8Xミトコンドリアバッファー：0.6 Mの最終濃度のスクロース10.28グラム、遊離脂肪酸の400mgのウシ血清アルブミン（BSA）0.8％、160 mMの最終濃度のためのHEPESの2.08グラム、pHの最終濃度蒸留水で50mlに7.4〜とQS。 </li><li>絶縁バッファ1（IB1は）：10に、215 mMの最終濃度のD -マンニトールの0.392グラム、最終濃度10 mM、500 mMのEDTA 200μlを加え8Xミトコンドリアバッファの1.25ミリリットル、7.4のpHとQS蒸留水とを添加した。 </li><li>絶縁バッファ2（IB2は）：10に、215 mMの最終濃度のD -マンニトールの0.392グラム、3 mMの最終濃度は500 mMのEGTAの60μlを加え8Xミトコンドリアバッファの1.25ミリリットル、7.4のpHとQS蒸留水とを添加した。 </li><li>実験的なバッファー（EB）：の最終濃度2.5mMのKH 2 PO 4を25μl、215 mMの最終濃度のD -マンニトールの0.392グラム、5 mMの最終濃度を500 mMのMgCl 2の100μlを追加する6.25μM、20μMの最終濃度を100 mMのEGTAを2μl、ミトコンドリアの緩衝液、pH7.4にし、QS蒸留水で10mlまでの1.25ミリリットル。 </li></ul></li></ol> 2。筋肉のアイソレーション<ol><li>アイスバケットでは、3つの10 mlのビーカー、ポッター-エルベージェム組織グラインダーと氷の上の楽器/消耗品その他のすべての必要を置く。すべては、実験を通して氷冷滞在する必要があります。 </li><li>行って温水槽でアイスバケットとEBのIB1とIB2を置きます。 </li><li> three 10ミリリットルのビーカーでは、以下のソリューションを追加する必要があります。 <ul><li>ビーカー1：PBSを10ml </li><li>ビーカー2：PBS / EDTAを10ml </li><li>ビーカー3：IB1 3 mlの</li></ul></li><li>人道的としてローカルの動物実験使用の委員会によって承認されたラットを殺す。 </li><li>急速に、骨格筋の250から500ミリグラムを分離ビーカー1で洗い流した後、ビーカー2に転送します。 </li></ol> 3。均質化/ミトコンドリア単離<ol><li>ビーカー3に筋肉を移し、細かくはさみで筋肉を飾らない。 </li><li>ポッター-エルベージェムホモジナイザーに解決策を転送する。電動乳棒（ドリルプレスの仕事をする）すべての回で氷の中にチューブを保持する10回を使って筋肉をホモジナイズする。 </li><li> 4℃で10分間、700 グラム ℃であらかじめ冷却した2 mlのマイクロチューブや遠沈管にホモジネート転送</li><li>新しい冷却した新しいマイクロチューブに上清を移す。ペレットを捨てる。 </li><li> 4℃で10分間、10500 グラムで上清を遠心℃に</li><li>新しい冷却した新しいマイクロチューブやラベルのSN1（清番号1）と筋肉の種類に上清を移す。 </li><li> IB2500μlにペレットを再懸濁する。 </li><li> 4℃で10分間、10500 グラムで遠心℃、 </li><li>新しい冷却した新しいマイクロチューブやラベルのSN2（清番号2）と筋肉の種類に上清を移します。 </li><li> IB2の100μlの最終的なミトコンドリアペレットを中断します。 </li><li>数秒間minifugeのリスピン最終ミトコンドリア懸濁液。ペレットがある場合は、新しい冷却した新しいマイクロチューブに上清を移すと、ペレットを捨てる。 </li><li>ブラッドフォードアッセイ8を使用してタンパク質濃度を決定する。 </li></ol> 4。電極のキャリブレーション注：ミトコンドリア分離時の完全な電極のキャリブレーション。 <ol><li> 250mlのフラスコに100mlの蒸留水を追加。大気中のガスと水を平衡化し、20分間激しく攪拌。 </li><li> Oxygraph電極にKClを50％の数滴を追加します。 </li><li>電極の上にブルーリズラ圧延紙の小片を置きます。 </li><li>ローリングペーパーの上にPTFEの膜の一部を置きます。 </li><li>リングのアプリケーターを用いて内輪を適用し、電極の溝に外輪を置きます。 </li><li>電極を接続し、機器の残りのアセンブル：大きなプラスチック製のリングのベース、ミトコンドリア室、および水のホースを。 </li><li> Oxygraphボックスをオンにし、Oxygraphのソフトウェアを起動します。 </li><li>ミトコンドリア室に平衡水を追加。 </li><li>攪拌棒を、60の速度にオン追加。 </li><li>新しい実験を始める。 </li><li>キャリブレーションをクリックし、ボックス1の液相のキャリブレーション。温度を37に変更° Cと&quot;OK&quot;を2回クリックします。 </li><li> &quot;OK&quot;と&quot;上書き&quot;の変更は、それをクリックし、窒素ガスのタンクを開くと。 </li><li>接続されている場所のヒントチャンバー内に窒素タンクへとゼロ酸素を確立する。それが切り替わる時、&quot;OK&quot;をクリックして&quot;上書き。&quot; </li><li>水を吸引し、ミトコンドリアのチャンバーへのEB緩衝液500μlを加える。 </li><li>プランジ​​ャーとシールチャンバー。 </li><li>ボックス2のキャリブレーションのため11月15日、複数のボックスは、手順を使用している場合。 </li></ol> 5。ミトコンドリア呼吸<ol><li>新しい実験を開始し、その信号を安定させるために約1分間実行してみましょう。 </li><li>各チャンバーに150μgのためにミトコンドリア懸濁液（ステップ3.11）の必要量、マークイベントを追加し、それが1分間実行してみましょう。 </li><li> 5 mM/2.5 mMの最終濃度のウォーム250 mM/125 mMのグルタミン酸/リンゴ酸を10μlを添加します。マークし、1分間実行してみましょう。これは、状態2のように定義されます。 </li><li> -150μMの最終濃度10 mMのADP7.5μlの、マークを、追加し、30秒間実行してみましょう。これは状態3と定義されています。 </li><li> 10 mMのADP、マークの別の7.5μlのを追加し、1.5分間実行してみましょう。 </li><li> 1μM、マークの最終濃度のために冷10 mMのオリゴマイシンの0.5μlを加え、3分間実行してみましょう。これは、状態4のように定義されます。 </li><li> 0.2μM、マークの最終濃度のコールド0.1mMのFCCPの1μlを加え、3分間実行してみましょう。これは、状態5として定義されています。 </li><li>終了したら、実験を終了して保存する。 </li><li> Oxygraphソフトウェアを使用して呼吸速度を取得する。 </li><li>正規化係数を入力してください：ミトコンドリアタンパク質150μgを添加すると、正規化係数は0.15となります。 </li><li>さまざまな呼吸状態のために正規化された呼吸速度を計算する。 </li><li>状態4によって状態3で割って呼吸調節率（RCR）を計算する。 </li></ol> 6。代表的な結果： <img src="/files/ftp_upload/2452/2452fig1.jpg" alt="図1" /> 図1は、。ミトコンドリア骨格筋での酸素消費量のトレース代表を示しています。電極信号が安定した後、ミトコンドリアサンプルはOxygraph室に追加されます。状態2呼吸は、グルタミン酸とリンゴ酸の添加により開始されます。 ADPの添加は、状態3呼吸を定義する、酸素消費量を増加させる。 ATP合成酵素は、状態4呼吸を得るためにオリゴマイシンを添加することによってブロックされます。最後に、FCCPはミトコンドリア呼吸（状態5）を切り離すために追加されます。 表1は、状態2、3、4、5の代表的酸素消費率を示しています。呼吸調節率（RCR）は、それぞれの実験のために計算されます。 RCR≥4は、実行可能なミトコンドリアの準備の証拠として考えられている。 <table border="1"><tbody><tr><td> 国 </td><td> 正規化されたレート </td></tr><tr><td>状態2 </td><td> 34.74 </td></tr><tr><td>状態3 </td><td> 153.40 </td></tr><tr><td>状態4 </td><td> 16.49 </td></tr><tr><td>状態5 </td><td> 143.70 </td></tr><tr><td> RCR </td><td> 9.30 </td></tr></tbody></table> 表1。ミトコンドリア呼吸速度。ミトコンドリア骨格筋からRepresentativeoxygen消費率。値は、チャンバーに追加されたミトコンドリアの量に正規化されます。呼吸調節率（RCR）が状態4によって状態3で割ることによって決定されます。 RCR≥4は、実行可能なミトコンドリアの準備を表します。

<ol>
	<li>Frezza, C., Cipolat, S., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+isolation:+functional+mitochondria+from+mouse+liver,+muscle+and+cultured+fibroblasts.">Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts.</a> Nat Protoc. 2, 287-295 (2007).</li><li>Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+mitochondria+in+health+and+disease.">Roles of mitochondria in health and disease.</a> Diabetes. 53, S96-S102 (2004).</li><li>Johannsen, D. L., Ravussin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mitochondria+in+health+and+disease.">The role of mitochondria in health and disease.</a> Curr Opin Pharmacol. , (2009).</li><li>Pallotti, F., Lenaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subfractionation+of+mitochondria+from+animal+cells+and+tissue+culture+lines.">Isolation and subfractionation of mitochondria from animal cells and tissue culture lines.</a> Methods Cell Biol. 80, 3-44 (2007).</li><li>Pallotti, F., Lenaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subfractionation+of+mitochondria+from+animal+cells+and+tissue+culture+lines.">Isolation and subfractionation of mitochondria from animal cells and tissue culture lines.</a> Methods Cell Biol. 65, 1-35 (2001).</li><li>Gamboa, J. L., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+content+and+distribution+changes+specific+to+mouse+diaphragm+after+chronic+normobaric+hypoxia.">Mitochondrial content and distribution changes specific to mouse diaphragm after chronic normobaric hypoxia.</a> Am J Physiol Regul Integr Comp Physiol. , (2009).</li><li>Patel, S. P., Gamboa, J. L., McMullen, C. A., Rabchevsky, A., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+respiratory+capacity+in+extraocular+muscle+mitochondria:+evidence+for+intrinsic+differences+in+mitochondrial+composition+and+function.">Lower respiratory capacity in extraocular muscle mitochondria: evidence for intrinsic differences in mitochondrial composition and function.</a> Invest Ophthalmol Vis Sci. 50, 180-186 (2009).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li></ol>

利害の衝突は宣言されません。

我々は、骨格筋から実行可能なミトコンドリアを分離するためのプロトコルを提示する。歩留まりに問題がある場合、プロトコルは氷中で30分間PBS/10mM EDTA/0.01％のトリプシン5mlに分離された筋肉をインキュベートすることによって変更することができます。トリプシンとの完全な筋肉の消化を確保するために、筋肉は完全にみじん切りする必要があります。 30分間のインキュベーション後、PB...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>95% CO2 / 5% O2 mix</td>
<td>&nbsp;</td>
<td>Local gas company</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine 5&#x2032;-diphosphate sodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A2754</td>
<td>&nbsp;</td>
<tr>
<td>Blue Rizla Paper</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>890101</td>
<td>&nbsp;</td>
<tr>
<td>Bradford protein assay</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>500-0006</td>
<td>&nbsp;</td>
<tr>
<td>Carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2920</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge 5804R</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Compressed nitrogen</td>
<td>&nbsp;</td>
<td>Local gas company</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>D-mannitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9647</td>
<td>&nbsp;</td>
<tr>
<td>Ethlyene-glycol-bis-tetraacetic acid (EGTA)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E3889</td>
<td>&nbsp;</td>
<tr>
<td>Ethylenediaminetetraacetic acid (EDTA)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>161-0728</td>
<td>&nbsp;</td>
<tr>
<td>Free fatty acid bovine serum albumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A8806</td>
<td>&nbsp;</td>
<tr>
<td>Glutamic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G5889</td>
<td>&nbsp;</td>
<tr>
<td>HEPES sodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H7006</td>
<td>&nbsp;</td>
<tr>
<td>Isotemp 3006D</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>&nbsp;</td>
<tr>
<td>Male Sprague Dawley Rats</td>
<td>&nbsp;</td>
<td>Harlan</td>
<td>300-500g</td>
<td>&nbsp;</td>
<tr>
<td>Malic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9138</td>
<td>&nbsp;</td>
<tr>
<td>Minifuge</td>
<td>&nbsp;</td>
<td>ISC Bioexpress</td>
<td>C1301P</td>
<td>&nbsp;</td>
<tr>
<td>Oligomycin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>O4876</td>
<td>&nbsp;</td>
<tr>
<td>Oxygen electrode disc</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>S1</td>
<td>&nbsp;</td>
<tr>
<td>Oxygraph</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Oxygraph Plus V1.01 Software</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>pH-meter</td>
<td>&nbsp;</td>
<td>Mettler Toledo</td>
<td>1225506149</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate-buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4417</td>
<td>&nbsp;</td>
<tr>
<td>Potassium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P3911</td>
<td>&nbsp;</td>
<tr>
<td>Potassium phosphate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P8416</td>
<td>&nbsp;</td>
<tr>
<td>Potter-Elvehjem homogenizers</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-414-14A</td>
<td>&nbsp;</td>
<tr>
<td>PTFE (0.0125mm &#215; 25mm) membrane</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>S4</td>
<td>&nbsp;</td>
<tr>
<td>SKIL 3320 drill press</td>
<td>&nbsp;</td>
<td>Hardware store</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S5016</td>
<td>&nbsp;</td>		</tbody>
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mitochondrial isolation from skeletal muscle

ミトコンドリア細胞の生と死を制御する細胞内小器官です。彼らは、主要な代謝反応に関与ATPの大部分を合成し、カスケード2,3をシグナリングの数を調整する。過去と現在の研究者は、肝臓、脳や心臓4,5としてラットやマウスの組織から単離ミトコンドリアいる。近年、多くの研究者は、骨格筋のミトコンドリア機能の研究に焦点を当てている。 ここで、我々は骨格筋6からミトコンドリアを単離するために正常に使用した方法を説明します。この場合の手続きは、すべてのバッファーと試薬が新鮮作られ、骨格筋の250から500まで約100mgを必要している必要があります。我々は、ラットとマウス腓腹筋と横隔膜から単離したミトコンドリアを研究し、ラットの眼の筋肉。ミトコンドリアタンパク質濃度はBradfordアッセイで測定されます。それは、ミトコンドリアのサンプルが準備中に氷冷状態に保つことと機能に関する研究が比較的短い時間（〜1時間）以内に測定することが重要です。ミトコンドリア呼吸は37クラーク型電極（Oxygraphシステム）° C 7でポーラログラフ分析を用いて測定されます。酸素電極のキャリブレーションは、このプロトコルの重要なステップであり、それを毎日実行する必要があります。単離ミトコンドリア（150μg）を実験的バッファー（EB）の0.5mlに追加されます。状態2呼吸は、グルタミン酸の添加（5mMの）とリンゴ酸（2.5 mM）で始まります。その後、アデノシン二リン酸（ADP）（150μM）は状態3を起動するために追加されます。オリゴマイシン（1μM）、ATPアーゼの酵素ブロッカーは、状態4を推定するために使用されます。最後に、カルボニルシアニドp型[トリフルオロメトキシが] -フェニル-ヒドラゾン（FCCP、0.2μM）は5 measurestateに追加、または呼吸6非結合される。呼吸調節率（RCR）、状態4から状態3の比率は、各実験後に計算されます。 RCR≥4は、実行可能なミトコンドリアの準備の証拠として考えられている。 要約すると、我々は、生化学的（例えば、酵素活性、免疫、プロテオミクス）と機能に関する研究（ミトコンドリア呼吸）で使用することができる骨格筋から実行可能なミトコンドリアの単離のための方法を提示する。

Watch this Scientific Journal Video about Mitochondrial Isolation from Skeletal Muscle at JoVE.com

Mitochondrial Isolation from Skeletal Muscle

このプロトコルは、骨格筋から単離したミトコンドリアの呼吸を研究するための手順を説明します。このメソッドは、Scorranoから翻案されました<em&gt;ら。</em&gt;（2007）。ミトコンドリアの単離方法は、約2時間を必要とします。ミトコンドリア呼吸は約1時間で完了することができます。

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 ...

mitochondrial-isolation-from-skeletal-muscle

Research

JoVE Journal

Biology

28.8K ビュー.  University of Kentucky. このプロトコルは、骨格筋から単離したミトコンドリアの呼吸を研究するための手順を説明します。このメソッドは、Scorranoから翻案されましたら。（2007）。ミトコンドリアの単離方法は、約2時間を必要とします。ミトコンドリア呼吸は約1時間で完了することができます。

ビデオ: Mitochondrial Isolation from Skeletal Muscle

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

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 ...

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 ...

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 ...

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.

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 and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

骨格筋線維芽細胞からのエクソソームの単離と特性評価

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

ビブラートムカット心筋切れ切り切れからヒト心室心筋細胞を分離

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

呼吸法アッセイのためのマウス骨格筋からのミトコンドリアの単離

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...