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>

<ol>
	<li>Brand, M., Nicholls, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+mitochondrial+dysfunction+in+cells.">Assessing mitochondrial dysfunction in cells.</a> Biochem. J. 435, 297-312 (2011).</li><li>Nicholls, D. G., Ferguson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioenergetics. , (2013).</li><li>Nunnari, J., Suomalainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+in+sickness+and+in+health.">Mitochondria: in sickness and in health.</a> Cell. 148, 1145-1159 (2012).</li><li>Lauri, A., Pompilio, G., Capogrossi, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+genome+in+aging+and+senescence.">The mitochondrial genome in aging and senescence.</a> Ageing Res. Rev. 18, 1-15 (2014).</li><li>Dube, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+acute+lipid+overload+on+skeletal+muscle+insulin+resistance,+metabolic+flexibility,+and+mitochondrial+performance.">Effects of acute lipid overload on skeletal muscle insulin resistance, metabolic flexibility, and mitochondrial performance.</a> Am. J. Physiol. Endocrinol. Metab. 12, 1117-1124 (2014).</li><li>Fernandez-Vizarra, E., Lopez-Perez, M. J., Enriquez, J. A. <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+biogenetically+competent+mitochondria+from+mammalian+tissues+and+cultured+cells.">Isolation of biogenetically competent mitochondria from mammalian tissues and cultured cells.</a> Methods (San Diego, Calif). 26, 292-297 (2002).</li><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>Rogers, G. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+microplate+respiratory+measurements+using+minimal+quantities+of+isolated+mitochondria.">High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria.</a> PloS one. 6, e21746 (2011).</li><li>Guarino, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+determining+oxygen+consumption+rates+of+static+cultures+from+microplate+measurements+of+pericellular+dissolved+oxygen+concentration.">Method for determining oxygen consumption rates of static cultures from microplate measurements of pericellular dissolved oxygen concentration.</a> Biotechnol. and Bioeng. 86, 775-787 (2004).</li><li>Will, Y., Hynes, J., Ogurtsov, V. I., Papkovsky, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mitochondrial+function+using+phosphorescent+oxygen-sensitive+probes.">Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes.</a> Nat. Protoc. 1, 2563-2572 (2007).</li><li>Hulver, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+stearoyl-CoA+desaturase-1+expression+in+skeletal+muscle+contributes+to+abnormal+fatty+acid+partitioning+in+obese+humans.">Elevated stearoyl-CoA desaturase-1 expression in skeletal muscle contributes to abnormal fatty acid partitioning in obese humans.</a> Cell Metab. 2, 251-261 (2005).</li><li>Frisard, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+4+modulates+skeletal+muscle+substrate+metabolism.">Toll-like receptor 4 modulates skeletal muscle substrate metabolism.</a> Am. J. Physiol. Endocrinol. Metab. 298, e988-e998 (2010).</li><li>Chance, B., Williams, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+respiratory+chain+and+oxidative+phosphorylation.">The respiratory chain and oxidative phosphorylation.</a> Adv. Enzymol. Relat. Subjects of Biochem. 17, 65-134 (1956).</li><li>Garcia-Cazarin, M. L., Snider, N. N., 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+isolation+from+skeletal+muscle.">Mitochondrial isolation from skeletal muscle.</a> JoVE. , (2011).</li><li>Gross, V. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+functional+mitochondria+from+rat+kidney+and+skeletal+muscle+without+manual+homogenization.">Isolation of functional mitochondria from rat kidney and skeletal muscle without manual homogenization.</a> Anal. Biochem. 418, 213-223 (2011).</li><li>Krieger, D. A., Tate, C. A., McMillin-Wood, J., Booth, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Populations+of+rat+skeletal+muscle+mitochondria+after+exercise+and+immobilization.">Populations of rat skeletal muscle mitochondria after exercise and immobilization.</a> J. Appl. Physiol.: Respir., Envir. and Ex. Physiol. 48, 23-28 (1980).</li><li>Asmann, Y. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+mitochondrial+functions,+mitochondrial+DNA+copy+numbers,+and+gene+transcript+profiles+in+type+2+diabetic+and+nondiabetic+subjects+at+equal+levels+of+low+or+high+insulin+and+euglycemia.">Skeletal muscle mitochondrial functions, mitochondrial DNA copy numbers, and gene transcript profiles in type 2 diabetic and nondiabetic subjects at equal levels of low or high insulin and euglycemia.</a> Diabetes. 55, 3309-3319 (2006).</li><li>Lanza, I. R., Nair, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assessment+of+isolated+mitochondria+in+vitro.">Functional assessment of isolated mitochondria in vitro.</a> Methods Enzymol. 457, 349-372 (2009).</li></ol>

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Essentially Fatty</td>
			<td rowspan="2">Sigma Aldrich</td>
			<td rowspan="2">A6003</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
			<td>Acid Free- BSA</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 
			White Crystals or Crystalline Powder 
			&#8805;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&#160;</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma Aldrich</td>
			<td>D6750&#160;</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 rowspan="2">Halt Protease &#38; Phosphatse Inhibitor Cocktail</td>
			<td rowspan="2">Thermo Scientific</td>
			<td rowspan="2">1861284</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">1.5mL microcentrifuge tubes with screw cap</td>
			<td rowspan="2">Thermo Scientific</td>
			<td rowspan="2">3474</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Zirconium Oxide beads</td>
			<td rowspan="2">Fisher Scientific</td>
			<td rowspan="2">C9012112</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</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 rowspan="2">Peroxidase conjugated affinipure Donkey, Anti Rabbit IgG (H+L)</td>
			<td rowspan="2">Jackson ImmunoResearh</td>
			<td rowspan="2">711-035-152</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Peroxidase conjugated affinipure Goat, Anti Mouse IgG (H+L)</td>
			<td rowspan="2">Jackson ImmunoResearh</td>
			<td rowspan="2">115-001-003</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</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&#160;</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&#176;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, &#8805;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 rowspan="2">L-Glutamic acid</td>
			<td rowspan="2">Sigma Aldrich</td>
			<td rowspan="2">G1251</td>
			<td rowspan="2">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>
		</tr>
		<tr>
			<td>Palmitoyl L-carnitine chloride</td>
			<td>Sigma Aldrich</td>
			<td>P1645</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Oligomycin A, &#8805; 95% (HPLC)</td>
			<td>Sigma Aldrich</td>
			<td>75351</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Carbonyl cyanide 4-(trifluoromethoxy)</td>
			<td rowspan="3">Sigma Aldrich</td>
			<td rowspan="3">C2920</td>
			<td rowspan="3">Store at 2-8&#176;C</td>
		</tr>
		<tr>
			<td>phenylhydrazone</td>
		</tr>
		<tr>
			<td>&#8805;98% (TLC), powder [FCCP]</td>
		</tr>
		<tr>
			<td>Antimycin A from streptomyces sp.</td>
			<td>Sigma Aldrich</td>
			<td>A8674</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td rowspan="2">Adenosine 5&#8242;-diphosphate monopotassium salt dehydrate [ADP]</td>
			<td rowspan="2">Sigma Aldrich</td>
			<td rowspan="2">A5285</td>
			<td rowspan="2">Store at -20&#176;C, to 7.4 with KOH prior to use in respirometric assay</td>
		</tr>
		<tr>
		</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

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 ...

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 ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

転写後調節される遺伝子の化学モジュレーターのためのハイスループットスクリーニング

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

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 High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Ca2+過渡現象とL型カルシウム電流の同時測定のための高品質マウス心房および心室筋細胞の分離

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions can be converted back to NO in mammals through a two-step reduction mechanism: nitrate being reduced to nitrite (NO2-) mostly by oral commensal bacteria, then nitrite being reduced to NO by several mechanisms including via heme- or molybdenum-containing proteins. This reductive nitrate pathway contributes to enhancing NO-mediated signaling pathways, particularly in the cardiovascular system and during muscular exercise. The levels of nitrate in the body before such utilization are determined by two different sources: endogenous NO oxidation and dietary nitrate intake, principally from plants. To elucidate the complex NO cycle in physiological circumstances, we have examined further the dynamics of its metabolites, nitrate and nitrite ions, which are relatively stable compared to NO. In previous studies skeletal muscle was identified as a major storage organ for nitrate ions in mammals, as well as a direct source of NO during exercise. Therefore, establishing a reliable methodology to measure nitrate and nitrite levels in skeletal muscle is important and should be helpful in extending its application to other tissue samples. This paper explains in detail the preparation of skeletal muscle samples, using three different homogenization methods, for nitrate and nitrite measurements and discusses important issues related to homogenization processes, including the size of the samples. Nitrate and nitrite concentrations have also been compared across four different muscle groups.

We present protocols for three different methods for the homogenization of four different muscle groups of rat skeletal muscle tissue to measure and compare the levels of nitrate and nitrite. Furthermore, we compare different sample weights to investigate whether tissue sample size affects the results of homogenization.

硝酸塩および亜硝酸塩測定のためのラット骨格筋ホモジネートの調製

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions ...