1。組織のコレクション： <ol><li> 7〜12週齢のマウスを使用してください。通常、少なくとも二つの動物が使用されています：一つは補償のために、蛍光マイナスワンアイソタイプコントロールサンプルのための必要なFACS単色のコントロールを設定することです。他には、ソートのための実際のサンプルです。 </li><li> CO 2吸入によりマウスを安楽死させるかの選択の倫理のプロトコルに従って。場所を上に向けてペーパータオルの上でマウス、およびマウスの毛皮で汚染を防ぐために、徹底的に70％エタノールでスプレー。 </li><li>鉗子による腹部の皮膚を持ち上げ、鋭いはさみと小さな縦方向のカットを行います。 </li><li>反対方向に皮膚の二つフラップ（上下）ピール完全に下に後肢の筋肉を公開する。 </li><li>脛骨の両側に沿ってはさみを挿入して拡張することによって消費税筋肉組織。 </li><li>筋肉組織を抽出するために大腿骨のための同じプロセスを繰り返します。 </li><li>各マウスからの組織のために一皿を使用して、60ミリメートルペトリ皿内のすべての摘出した筋肉組織を置きます。 </li><li>すべてのマウスは、上記を繰り返します。 </li></ol> 2。酵素消化によって単一細胞に組織の関連付けを解除します。 プロトコルのこのセクション全体で、滅菌試薬およびツールを使用し、無菌条件下で動作する。 <ol start="9"><li>各ペトリ皿にコラゲナーゼIIの2 mLの（シグマ、カタログ番号6885、500U / mL）および250mmのCaCl 2の株式の20μLを追加。 </li><li>非常に汚染されたとして削除し、破棄する注意を払って、二つの鉗子（一鋸歯状、ファイン科学ツールから2歯を持つもの、、猫＃11051から10、11154〜10）の小さな断片（約1mm 3）に筋肉組織を引き裂く可能な限り、非筋肉組織。 </li><li>ペトリ皿を覆い、30分間37℃でそれらをインキュベートする。 </li><li>ペトリ皿を取得し、徹底的にマッシュ組織片に3 mLシリンジのプランジャーを使用してください。汚染を避けるために、サンプルごとに1つのプランジャーを使用してください。 </li><li>各ペトリ皿にいくつか（〜5mL）を滅菌冷PBSを追加し、（必要なすべての組織を回復する場合ペトリ皿を洗う）50mLのファルコンチューブに組織の汚泥を転送する</li><li>精力的にファルコンチューブを振る、PBSでチューブを埋める、遠心分離機@約800 rpm × 5分（エッペンドルフのベンチトップモデル：5810R）、そして上清を吸引（繰り返し3回） </li><li> 37℃でインキュベートし、各チューブにし、CaCl 2の10μL（250MM）：1mLのコラゲナーゼDの混合物（ロシュ、カタログ番号11088882001、1.5U / mL）を/ディスパーゼII（04942078001、2.4u / mLのロシュ、猫＃）を追加する1時間回転で摂氏。 </li><li>冷たい、滅菌PBS、渦と組織を均質化するピペットの5-10 mLを加え。 </li><li> PBSでチューブをいっぱいにしてよく混ぜる。 </li><li>組織の残りの大部分を除外するために50mlのファルコンチューブの上に配置さ40μmのセルストレーナーに液体を移す。均等にthree 50mLのファルコンチューブに各サンプルを分ける。 PBSで各チューブを埋めるには、摂氏8度で5分間@ 1600rpmを遠心してください。 </li><li>上清を捨て、染色用の細胞を収集する。 </li></ol> 3。抗体染色およびソート： プロトコルのこのセクション全体で、無菌のFACSおよびコレクションのバッファを使用し、無菌条件下で動作する。適切に取り扱えば商業抗体は一般に、無菌と考えられている。 <ol start="20"><li>単一の色やアイソタイプコントロールサンプルの場合は、1mLのFACS緩衝液でサンプルを再懸濁し、9つの1.5mlのプレ標識したエッペンドルフチュー​​ブ（100μLを各試験管）に懸濁した細胞を分ける。 <ol><li>単一の色の染色を設定し、次の表にしたがってミックス。 注意：染色のアイソタイプコントロール抗体の終濃度は、一次抗体の彼らはのための制御であることと一致していなければなりません。抗Sca - 1が1μg/106セルに使用されている場合例えば、、その対応するアイソタイプは、同じ濃度で使用されている必要があります。 <table border="1"><tbody><tr><td>チューブ＃ </td><td>チューブの名前</td><td>会社</td><td>猫＃ </td><td>クローン</td><td>アイソタイプ</td><td>希釈</td></tr><tr><td> 1 </td><td>非染色</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 2 </td><td> Hoechst33342 </td><td>シグマ</td><td> B2261 </td><td></td><td></td><td> 2-5ug/ml </td></tr><tr><td> 3 </td><td> PI </td><td>シグマ</td><td> P4864 </td><td></td><td></td><td> 1ug/ml </td></tr><tr><td rowspan="2"> 4 </td><td rowspan="2"> CD31 - FITC CD45 - FITC </td><td>ベイバイオサイエンス</td><td> 11-0311-85 </td><td> 390 </td><td>ラットIgG2a </td><td> 500 </td></tr><tr><td>ハウスは作ら</td><td></td><td> I3 / 2 </td><td>ラットIgG2b </td><td> 500 </td></tr><tr><td> 5 </td><td> SCA1 - PECY7 </td><td>ベイバイオサイエンス</td><td> 25-5981-82 </td><td> D7 </td><td>ラットIgG2a </td><td> 5000 </td></tr><tr><td> 6 </td><td> α- 7 APC </td><td>ハウスは作ら</td><td></td><td> R2F2 </td><td>ラットIgG2b </td><td> 1000年</td></tr></tbody></table></li><li>アイソタイプコントロール染色には、次の表を使用して混在させる設定します。 <table border="1"><tbody><tr><td>チューブ＃ </td><td>の名前 チューブの</td><td>ヘキスト</td><td> PI </td><td> CD31 - FITC </td><td> CD45 - FITC </td><td> SCAL - PE - CY7 </td><td> -7 - APC </td><td>ラット、 IgG2b - APC </td><td>ラット、 的なIgG2a - pecy7 </td><td>ラット、 的なIgG2a - FITC </td><td>ラット、 IgG2b - FITC </td></tr><tr><td> 7 </td><td> ISO - CD31 / CD45 </td><td> + </td><td> + </td><td> - </td><td> - </td><td> + </td><td> + </td><td> - </td><td> - </td><td> + </td><td> + </td></tr><tr><td> 8 </td><td> ISO - SCAL - pecy7 </td><td> + </td><td> + </td><td> + </td><td> + </td><td> - </td><td> + </td><td> - </td><td> + </td><td> - </td><td> - </td></tr><tr><td> 9 </td><td> ISO - α- 7 - APC </td><td> + </td><td> + </td><td> + </td><td> + </td><td> + </td><td> - </td><td> + </td><td> - </td><td> - </td><td> - </td></tr></tbody></table></li><li>ステップ20から適切なエッペンドルフチュー​​ブにピペットで単一色と同位体制御の染色ミックスを（我々は通常ミックスを染色の5〜10μLを各サンプルに追加されているので、抗体の濃度を調整する）。よく混和して、1時間氷上でインキュベートする。 </li></ol></li><li> FACSの細胞選別のための抗体のカクテル（下）の800μLを持つサンプルのマウスから細胞を染色。よく混和して、1時間氷上でインキュベートする。 抗体のカクテル：ヘキスト、PI、CD31 - FITC、CD45 - FITC、SCA1 - PECY7、α- 7 APC 注意：重要な違いがバッチ間に存在する可能性があるため、細胞への抗体の最適比は、個別に各抗体を滴定によって決定される必要があります。使用する抗体の最適量は10 6の標的細胞当たりの抗体のμgsで発現し、抗体の同じバッチを使用する際に一定に保つ必要があります。明らかに、標的細胞の数は、実験間で著しく変化しない場合、または染色液は細胞の数を拡大縮小する場合、各抗体の固定希釈にも使用することができます。 </li><li>洗って：9コントロールチューブの各々にFACS緩衝液の約1mLの追加、サンプルチューブにFACS緩衝液の約20mlのを追加。 </li><li> 5分間エッペンドルフチュー​​ブ@ 3000rpmのを遠心（エッペンドルフベンチトップ遠心機、モデル：5417C）。サンプルチューブを5分間@ 1600rpm（：5810Rエッペンドルフのベンチトップ遠心機、モデル）心する。 </li><li>すべてのチューブから上清を吸引して捨てます。 </li><li> FACS緩衝液（コントロールの各1mLの、ソートサンプルの4mlの）に再懸濁し、細胞は、&quot;FACSチューブ&quot;セルストレーナーキャップを通して（BDファルコンの猫＃352235、40μmの孔径）にピペットの細胞は、本資料の残りの塊を除去するサイトメーターに影響を及ぼす。 </li><li>単一のカラーコントロールおよびアイソタイプコントロールによるFACSソーターを設定します。 </li><li>別々に3.5mLの収集バッファー（95％DMEM、5％FBS）で、次の表にしたがってソートされた細胞を集めます。通常は、両方の後肢を収穫するときに、単一の野生型のマウスは95％以上の生存率で、15万筋前駆細胞または100,000脂肪細胞前駆細胞について得られることができる、などの手続きの終わり（図1をFACSで再測定し選別された細胞で判断） 表面染色に基づいて、筋原性と脂肪細胞前駆細胞の定義 <table border="1"><tbody><tr><td>抗体/染色</td><td>筋原性前駆細胞（MP） </td><td>脂肪生成前駆細胞（AP） </td></tr><tr><td>ヘキスト</td><td> + </td><td> + </td></tr><tr><td> PI </td><td> - </td><td> - </td></tr><tr><td> CD31 - FITC </td><td> - </td><td> - </td></tr><tr><td> CD45 - FITC </td><td&gt; - </td><td> - </td></tr><tr><td> SCA1 - PECY7 </td><td> - </td><td> + </td></tr><tr><td> α- 7 APC </td><td> + </td><td> - </td></tr></tbody></table></li></ol> 4。移植： <ol start="28"><li> @ 1500RPMで5分間、オートクレーブエッペンドルフチュー​​ブ（1.5 mL）に上清および転送細胞を除去ソートした細胞を遠心、5分間@ 3000rpmのを遠心分離し、滅菌PBS（血清フリー！）の500μLで細胞をリンス。滅菌条件下で、すべての試薬 ​​を維持し、組織培養フードで働いて、約10 6細胞/ mLのPBSで筋原性前駆細胞、またはマトリゲルで脂肪細胞前駆細胞（BDの猫＃354234）を懸濁します。 </li><li>レシピエントマウスに筋原性前駆細胞または脂肪細胞前駆細胞を移植。 <ol><li> isoflouranはあなたの機関のポリシーにしたがってマウスをAnaesthetize。 </li><li>注入領域の周りの毛を剃る、その後70％エタノールでこすって皮膚を消毒する</li><li> 3月10日CCのインスリン注射器（BDの猫＃309300）を使用して、筋原性前駆細胞または脂肪細胞前駆細胞（= 20K細胞）の20μLを注入する。 </li></ol></li><li>適切な時間（3週間も動作しますが、ドナー由来のトランスジェニックマーカーを発現する筋線維は、移植後一週間以内に通常明らかである）の後に、（Sigmaカタログ＃T04840 - 2、400μLAVERTINの腹腔内注射することにより、ステップ29からのレシピエントマウスをanaesthetize 25 mg / mL）は、その後、4％PFAの15mlのに続いて50mLのPBS（EDTAの10μMを含む）最初の、とtranscardially灌流。必要に応じて、一晩20％ショ糖に転送し、一晩4％PFAで標的組織、ポストフィックスを収集する。 OCT、凍結および凍結切片に埋め込みます。 </li></ol> 5。代表的な結果： MPがマウス骨格筋に移植されると、彼らは容易に既存の筋線維と融合。したがって、ドナー細胞中に存在する遺伝子のラベルは、それを受信した繊維で容易に検出可能となります。図2に移植された細胞は、ヒトアルカリphostphataseを発現する組織化学的に明らかにした例を示しています。 APが移植されている場合、結果は移植部位の環境に大きく依存しています：（図2参照）これらの細胞は脂肪細胞や筋線維芽細胞に起源を与える皮下移植したとき。脂肪変性は3を誘導されていない限り、筋肉を含む他の多くのサイトで、で、彼らは生きていけない。 <img src="/files/ftp_upload/2476/2476fig1.jpg" alt="図1" /> 図1。骨格筋からの前駆細胞集団の分離のための戦略をソート （）戦略ソート：生存細胞は前方散乱と側方散乱に基づいて同定されたが。ヘキスト染色は、死細胞を除外するために無核の残骸とヨウ化プロピジウム（PI）染色を除外するために使用されました。造血（CD45）と内皮（CD31）の細胞をソーティングゲートから除外した。 α7+ヘキスト+ PI - CD45 - CD31 - SCAL -サブセットは、すべての筋原性前駆細胞（MP）が含まれています。 SCAL +ヘキスト+ PI - CD45 - CD31 - α7 -人口は、脂肪細胞前駆細胞（AP）が含まれています。 （B）蛍光マイナスワン（FMO）アイソタイプコントロール染色の特異性を確認する。 （C）純度は、ソート後のMPとAPのサブセ​​ットをチェックします。 <img src="/files/ftp_upload/2476/2476fig2.jpg" alt="図2" /> 図2。 MPとAP移植後の代表的な結果。APは、皮下に移植（上部パネル）とMP筋肉内（下部パネル）された。どちらの場合も、ドナー細胞は、茶色の染色によって同定された遺伝子組換えヒトアルカリホスファターゼを発現するマウス由来。

<ol>
	<li>Buckingham, M., Montarras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+stem+cells.">Skeletal muscle stem cells.</a> Curr Opin Genet Dev. 18, 330-336 (2008).</li><li>Relaix, F., Marcelle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells.">Muscle stem cells.</a> Curr Opin Cell Biol. 21, 748-753 (2009).</li><li>Joe, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+injury+activates+resident+fibro/adipogenic+progenitors+that+facilitate+myogenesis.">Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.</a> Nat Cell Biol. 12, 153-163 (2010).</li><li>Natarajan, A., Lemos, D. R., Rossi, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibro/adipogenic+progenitors:+A+double-edged+sword+in+skeletal+muscle+regeneration.">Fibro/adipogenic progenitors: A double-edged sword in skeletal muscle regeneration.</a> Cell Cycle. 9, (2010).</li><li>Sherwood, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+adult+mouse+myogenic+progenitors:+functional+heterogeneity+of+cells+within+and+engrafting+skeletal+muscle.">Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle.</a> Cell. 119, 543-554 (2004).</li><li>Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+and+expansion+of+single+transplanted+muscle+stem+cells.">Self-renewal and expansion of single transplanted muscle stem cells.</a> Nature. , (2008).</li><li>Montarras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+isolation+of+satellite+cells+for+skeletal+muscle+regeneration.">Direct isolation of satellite cells for skeletal muscle regeneration.</a> Science. 309, 2064-2067 (2005).</li></ol>

利害の衝突は宣言されません。

このプロトコルの目的は、高収率および精製した細胞の高い生存の間に合理的なバランスを取ることです。健康な細胞を回収されていることを確保する上で最も重要なステップは、予想通り、出発組織の酵素的解離です。組織の取扱いは、デモンストレーションや視聴覚の形式でも鑑賞するのは困難である、特に穏やかなはずです。もう一つの重要な要素は、プロシージャの長さです。もはやそれは、したがって、より低い生存率と、生着効率、ドナーからレシピエント動物に移動されます。すぐにソートした後、精製した細胞サンプル中のPI肯定的な出来事の頻度を見ることによって応答されていない実行可能性についての質問があるはず、私たちはclonogenicityを測定するために限界希釈アッセイは3を実行されることを示唆している。無傷の筋肉からの典型的なソートは、日常的にin vitroでの筋原性または線維/脂肪細胞コロニーを開始することのできる15から20まで細胞内で約1が得られる。国会議員から開始されたコロニーの本質的に100％は差別化された筋管を含んでいますが、FAPSから得られたコロニーの約3分の1は、平滑筋アクチン正の筋線維芽細胞に加えて脂肪細胞が含まれています。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td>コラゲナーゼII </td><td>シグマアルドリッチ</td><td> C - 6885、 </td><td> 500 U / mlの</td></tr><tr><td>コラゲナーゼD </td><td>ロッシュ</td><td> 11088882001 </td><td> 1.5 U / mlの</td></tr><tr><td> IIを置き換える</td><td>ロッシュ</td><td> 04942078001 </td><td> 2.4 U / mlの</td></tr><tr><td>セルストレーナー</td><td> BD </td><td> 352340 </td><td> 40μmの</td></tr><tr><td>セルストレーナー付きチューブ</td><td> BD </td><td> 352235 </td><td> 40μmの</td></tr><tr><td> Hoechst33342 </td><td>シグマアルドリッチ</td><td> B2261 </td><td> 2月5日ug / mlと</td></tr><tr><td> CD31 - FITC </td><td> Ebiosciences </td><td> 11-0311-85 </td><td>クローン390 </td></tr><tr><td> CD45 - FITC </td><td>ホームメイド</td><td></td><td>クローンI3 / 2 </td></tr><tr><td> SCA1 - PECY7 </td><td> Ebiosciences </td><td> 25-5981-82 </td><td>クローンD7 </td></tr><tr><td> α- 7インテグリンAPC </td><td>ホームメイド</td><td>ロッシラボにお問い合わせ</td><td> R2F2 </td></tr></tbody></table>

purification of progenitors from skeletal muscle

骨格筋は、別個の胚起源と発達の可能性の複数の前駆細胞集団が含まれています。筋原性前駆細胞、通常ensheathsそれは、単に筋系統の1,2にコミットしている自己複製細胞であること。筋線維形質膜とラミニンが豊富な基底膜の間に&quot;人工衛星細胞の位置&quot;に常駐している我々は最近、効率的に増殖を入力して、損傷に応答し、組織再生3,4中に筋原細胞への栄養サポートを提供する筋線維芽細胞と白色脂肪細胞を生成することができる容器関連する前駆細胞、第二のクラスを記載している。特定の細胞集団の発達と再生の可能性を判断する最も信頼されるアッセイの一つは、彼らの分離および5-7移植に依存しています。この目的のために我々はこの記事で概説する酵素的に解離した筋肉からのフローサイトメトリー、で、その精製のためのプロトコルを最適化してきました。この方法を用いて得られた個体群は、筋原性または線維/脂肪細胞コロニー形成細胞のいずれかが含まれます：他の細胞型は、in vitroで拡大またはin vivoでの配信で生き残ることができるではありません。ただし、これらの集団は、1つは、新しくソートされたサブセットは、コロニーを形成するか、受信者の生着の能力を欠いている他の汚染物質の細胞を含んでいる可能性があることに留意する必要が分子解析のためのソート（例えば定量RT - PCR）の直後に使用されています。

Watch this Scientific Journal Video about Purification of Progenitors from Skeletal Muscle at JoVE.com

Purification of Progenitors from Skeletal Muscle

酵素的解離、線維/脂肪細胞とマウス骨格筋からの筋原性前駆細胞のフローサイトメトリーによる表面標識および精製方法。

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 ...

purification-of-progenitors-from-skeletal-muscle

Research

JoVE Journal

Biology

18.2K ビュー.  University of British Columbia. 酵素的解離、線維/脂肪細胞とマウス骨格筋からの筋原性前駆細胞のフローサイトメトリーによる表面標識および精製方法。

ビデオ: Purification of Progenitors 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

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 ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

生細胞におけるストア作動性カルシウムエントリの蛍光ベースの測定：培養癌細胞から骨格筋線維に

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

ゼブラフィッシュ胚骨格筋のレーザー招いた傷害

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

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 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 ...

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

ラットの骨格筋における線維-刺激性前駆腫(FAPs)および筋原性前駆腫(MP)の同定、分離、および特徴付け

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

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 ...

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.