この研究は、国立科学工学研究協議会 (レベル、探索許可) およびユニヴェルシテ ・ ド ・ モンクトンから NP に補助金によって賄われていた。1塩基変異は、健康研究 (機構)、ニュー ・ ブランズウィック州イノベーション財団 (NBIF) およびユニヴェルシテ ・ ド ・ モンクトンのカナダの研究所から資金調達のサポートを確認したいと思います。EHC の作業をサポートするには、カナダ、脳カナダ、レベル、カナダ乳がん基金、ニュー ・ ブランズウィック州技術革新基金、ニューブランズウィックの健康研究財団のアルツハイマー協会とユニヴェルシテ ・ ド ・ モンクトン。

1. 試薬の調製
<ol><li>解剖と組織の透過の以下のソリューションを準備します。<ol>
<li>保存液を準備: 2.77 ミリメートル CaK27.23 mM K2グリコールエーテルジアミン四酢酸、5.77 mM Na2ATP、6.56 mM MgCl2、タウリン、20 mM 15 mM Na2クレアチンリン酸、20 mM のイミダゾール、0.5 mM ジチオトレイトール グリコールエーテルジアミン四酢酸と 50 ミリメートル K-MES の pH 7.1 (格納できます。-20 ° c)。</li>
		<li>サポニン溶液の準備: 保存液の 1 mL のサポニンの 5 mg (新鮮な毎日を準備)。</li>
	</ol>
</li>
	<li>呼吸の測定のための次のソリューションを準備します。<ol>
<li>呼吸メディアの準備: 120 mM KCl、5 mM KH2PO4、3 mM HEPES、1 mM グリコールエーテルジアミン四酢酸、1 mM MgCl2、および 0.2 %bsa (w/v)、pH 7.2。</li>
		<li>基板、ランプ、阻害剤を準備します。H2O、すべての基板を溶解し、エタノール (H2O で希釈したマロン酸) を除いて絶対に阻害剤と同様、FCCP ランプを解散します。ほとんど、基板と同様のマロン酸 (材料の表を参照) を中和します。プロトコルは、すべての濃度は、2 mL 槽内最終濃度です。</li>
	</ol>
</li>
</ol>2. 準備中と高解像度計のキャリブレーション。
<ol><li>洗浄室、キャップ、および蒸留水 70% エタノールで 3 回と 3 回の呼吸のストッパー。</li>
	<li>ゼロ酸素濃度調整: チャンバ内呼吸媒体の 2.3 mL ピペットで移しなさい、亜ジチオン酸ナトリウム 10 mg を追加。商工会議所の最大の量は 2 mL が、2.3 mL は、維持気泡なし、ストッパーの下チャンバーを閉じるときに追加されます。ストッパーで部屋を閉じ、酸素濃度が 10-20 分調整結果の酸素消費量と酸素濃度がゼロで電極を測定します。</li>
	<li>芳醇な水で部屋を洗って 10 分、無水エタノールで孵化させなさい蒸留水で洗い流し。</li>
	<li>空気飽和状態における調整: ピペット 2.3 mL 呼吸室、中のストッパーを挿入、ストッパーの上に余分な培地を吸引、スペーサー、ストッパーを持ち上げます。戻過剰呼吸媒体は、部屋を閉じた後、気泡の存在を避けるために室 (合計 2 mL の体積) で。</li>
	<li>酸素濃度 (ブルー トレース) が 250 前後安定するまで 1 時間 45 分の酸素消費量を監視 24 の ° C で nM</li>
</ol>3. ハエや組織の透過の郭清
<ol><li>氷の上のすべての手順を実行していることを確認します。</li>
	<li>同じ系統の六つの男性のハエを anaesthetize、同じ年齢、同じ食事 (野生型 w1118, 15 日齢、コーンミール培地に供給) ハエの解離を容易にするために氷の上で発生します。</li>
	<li>飛翔筋を含む thoraxes のみを維持する (頭と腹部を削除) ハエを解剖メスと細い鉗子を使用する。この手順は、肉眼で行うことができます。先の細いピンセットで結果の thoraxes を処理します。</li>
	<li>25 mm シャーレ、鉗子を用いた氷冷保存液 2 mL を含む 3 つの解剖 thoraxes を転送します。</li>
	<li>先の細いピンセットで機械的に鉗子の先端を挿入、thoraxes、thoraxes を permeabilize し、繰り返し緩く接続されたネットワークを取得する組織を引き裂きます。</li>
	<li>24 ウェル プレートでサポニン溶液と、サポニンの 62.5 μ G/ml の濃度が最終的な保存液 1 mL 12.5 μ L で 2 つの井戸を埋めます。呼吸中の 1 mL の 2 つの隣接する井戸を埋めます。</li>
	<li>先の細いピンセットで希釈したサポニン溶液に 3 グリセリン thoraxes を転送し、軌道シェーカーで、氷の上に 20 分間の軽度の動揺と孵化します。</li>
	<li>後者のインキュベーション後軽度の動揺、サポニンを洗うこと 5 分間氷の上で呼吸中でいっぱい隣接する井戸に繊維を転送します。</li>
</ol>4. 乾燥減量の定量
<ol><li>グリセリン thoraxes 吸収性の表面上の乾燥し、3-4 回はすべての水分が除去されるかどうかを確認する細い鉗子を使用してフリップします。</li>
	<li>発振に一緒に 3 つの thoraxes の重量を量る。ミトコンドリアの酸素消費量が正規化される、得られた重みに注意してください。</li>
	<li>氷の上、呼吸中のドロップですぐに組織を転送します。</li>
</ol>5. 酸素消費率の定量
<ol><li>呼吸度計の室を開く (ストッパーを削除) し、各チャンバーに 10 mM ピルビン酸とリンゴ酸の 2 mM を追加。</li>
	<li>呼吸の中で満たされた室に直接グリセリン組織を追加します。</li>
	<li>スペーサーを使用してストッパーを取り付けます。</li>
	<li>60 mL シリンジと酸素タンクから直接酸素を収集し、組織中の酸素拡散が酸素消費量を制限していないかどうかを確認する各商工会議所のストッパー毛細血管を通して酸素の 2-5 mL を注入します。</li>
	<li>商工会議所に、thoraxes が残っているかどうかを確認する各ストッパーの毛細血管にハミルトン シリンジを挿入します。</li>
	<li>酸素濃度が約 400 室を完全に閉じる空気飽和上の酸素のレベルを達成する nM (約 160% は飽和空気) 部屋の中。</li>
	<li>停止し、チャンバ内の空気の泡を確認するミキサーを再起動します。</li>
	<li>組織呼吸の組織 (mg) の質量質量固有の酸素消費量 (pmol O2 consumed.s-1.mg-1組織) の表示を正常化する以前の重量を量った量を入力します。</li>
	<li>酸素消費量が (赤いトレース) を安定して、5 mM ADP ハミルトン注射器を追加します。</li>
	<li>各注入の後 70% エタノール蒸留水と注射器を洗い、再び水を蒸留します。</li>
	<li>酸素消費量が再び安定、シトクロム c の 4 mM の 5 μ L を注入します。</li>
	<li>ETS のさまざまなステップでミトコンドリアの酸素消費量を評価する次の基板の連続注射を行います。<ol>
<li>2 M のプロリンの 5 μ L を追加します。</li>
		<li>1 M のコハク酸の 10 μ L を追加します。</li>
		<li>1 M グリセロール-3-リン酸の 30 μ L を追加します。</li>
	</ol>
</li>
	<li>0.5-1 の手順で滴定方法でランプ FCCP を注入 μ M (1 mM の溶液 2 μ L) 最適な濃度までは達された、すなわち、抑制することがなく可能な限り最高の酸素消費量を達成するために濃度 (注意:テーブルを参照してください材料の)。</li>
	<li>完全 (ロックス) の残留酸素消費を測定するために ETS の電子流束を阻害する順番に次の阻害剤の注入など他の中ヘムオキシゲナーゼ反応すなわち非呼吸反応。<ol>
<li>1 mM ロテノンの 1 μ L を追加 (注意:材料の表を参照してください)。</li>
		<li>2 M のマロン酸の 5 μ L を追加 (注意:材料の表を参照してください)。</li>
		<li>5 mM アンチマイシン A の 1 μ L を追加 (注意:材料の表を参照してください)。</li>
	</ol>
</li>
	<li>アスコルビン酸 0.2 mM や 0.5 mM 受けた東京都警視庁、室に順次複雑な IV による酸素消費量を測定するアスコルビン酸で始まるを追加します。</li>
	<li>複雑な IV を阻害するアジの 20 mM を追加 (注意:材料の表を参照してください)。</li>
	<li>信号が安定して、チャンバー スペーサーで、純粋な酸素の 2 mL を注射することによって部屋を再酸素開閉チャンバー濃度が 250-300 nmol.mL-1。</li>
	<li>10-15 分のための信号を測定し、化学の背景、すなわち、受けた東京都警視庁の自動酸化による酸素消費量を計算します。</li>
</ol>6. 工場の洗浄
<ol><li>測定後蒸留水でチャンバーを濯ぎ 1 回と 3 回 70% のエタノールのチャンバーをすすいでください。</li>
	<li>阻害物質を不活化する 10 分の無水エタノールで孵化させなさい。</li>
	<li>蒸留水すすぎ 3 回、70% エタノールで部屋を埋める、次利用までストッパーとキャップを交換します。</li>
</ol>

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D., Nicholls, D. G. <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> Biochemical Journal. 435 (2), 297-312 (2011).</li><li>St-Pierre, J., Buckingham, J. A., Roebuck, S. J., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topology+of+superoxide+production+from+different+sites+in+the+mitochondrial+electron+transport+chain.">Topology of superoxide production from different sites in the mitochondrial electron transport chain.</a> Journal of Biological Chemistry. 277 (47), 44784-44790 (2002).</li><li>Kuznetsov, A. V., Veksler, V., Gellerich, F. N., Saks, V., Margreiter, R., Kunz, W. S. <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+in+situ+in+permeabilized+muscle+fibers,+tissues+and+cells.">Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells.</a> Nature Protocols. 3 (6), 965-976 (2008).</li><li>Gnaiger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacity+of+oxidative+phosphorylation+in+human+skeletal+muscle.+New+perspectives+of+mitochondrial+physiology.">Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology.</a> International Journal of Biochemistry and Cell Biology. 41 (10), 1837-1845 (2009).</li><li>Picard, M., Taivassalo, T., Gouspillou, G., Hepple, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+isolation,+structure+and+function.">Mitochondria: isolation, structure and function.</a> The Journal of Physiology. 589 (18), 4413-4421 (2011).</li><li>Pichaud, N., Ballard, J. W. O., Tanguay, R. M., Blier, P. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+sensitivity+of+mitochondrial+functions+in+permeabilized+muscle+fibers+from+two+populations+of+Drosophila+simulans+with+divergent+mitotypes.">Thermal sensitivity of mitochondrial functions in permeabilized muscle fibers from two populations of Drosophila simulans with divergent mitotypes.</a> American Journal of Physiology - Regulatory, Integrative and Comparative Physiology. 301 (1), R48-R59 (2011).</li><li>Pichaud, N., Ballard, J. W. O., Tanguay, R. M., Blier, P. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+occurring+mitochondrial+dna+haplotypes+exhibit+metabolic+differences:+insight+into+functional+properties+of+mitochondria.">Naturally occurring mitochondrial dna haplotypes exhibit metabolic differences: insight into functional properties of mitochondria.</a> Evolution. 66 (10), 3189-3197 (2012).</li><li>Pichaud, N., Messmer, M., Correa, C. C., Ballard, J. W. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet+influences+the+intake+target+and+mitochondrial+functions+of+Drosophila+melanogaster+males.">Diet influences the intake target and mitochondrial functions of Drosophila melanogaster males.</a> Mitochondrion. 13 (6), 817-822 (2013).</li><li>Wolff, J. N., Pichaud, N., Camus, M. F., C&#244;t&#233;, G., Blier, P. U., Dowling, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+implications+of+mitochondrial+genetic+variation:+mitochondrial+genetic+effects+on+OXPHOS+respiration+and+mitochondrial+quantity+change+with+age+and+sex+in+fruit+flies.">Evolutionary implications of mitochondrial genetic variation: mitochondrial genetic effects on OXPHOS respiration and mitochondrial quantity change with age and sex in fruit flies.</a> Journal of Evolutionary Biology. 29 (4), 736-747 (2016).</li><li>Gnaiger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacity+of+oxidative+phosphorylation+in+human+skeletal+muscle.">Capacity of oxidative phosphorylation in human skeletal muscle.</a> The International Journal of Biochemistry &amp; Cell Biology. 41 (10), 1837-1845 (2009).</li><li>Phang, J. M., Donald, S. P., Pandhare, J., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metabolism+of+proline,+a+stress+substrate,+modulates+carcinogenic+pathways.">The metabolism of proline, a stress substrate, modulates carcinogenic pathways.</a> Amino Acids. 35 (4), 681-690 (2008).</li><li>Bender, T., Martinou, J. 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+pyruvate+carrier+in+health+and+disease:+To+carry+or+not+to+carry?.">The mitochondrial pyruvate carrier in health and disease: To carry or not to carry?.</a> Biochimica et Biophysica Acta - Molecular Cell Research. 1863 (10), 2436-2442 (2016).</li><li>Divakaruni, A. 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=Thiazolidinediones+are+acute,+specific+inhibitors+of+the+mitochondrial+pyruvate+carrier.">Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (14), 5422-5427 (2013).</li><li>McCommis, K., 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=An+ancestral+role+for+the+mitochondrial+pyruvate+carrier+in+glucose-stimulated+insulin+secretion.">An ancestral role for the mitochondrial pyruvate carrier in glucose-stimulated insulin secretion.</a> Molecular Metabolism. 5 (8), 602-614 (2016).</li></ol>

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本研究では、ショウジョウバエにおけるミトコンドリアの酸素消費量の測定前の試料の調製方法を説明します。このメソッドは、期間、必要な人数の面で特に、ミトコンドリアの分離を使用してプロトコルに関連するさまざまな問題を克服するために開発されました。通常いくつかの個人から得られた組織の大規模な量を必要とするミトコンドリアの分離ではなく、この実験はいくつかのシ?...

ミバエショウジョウバエは、1世紀以上の遺伝子研究のモデル生物として使用されています。この生物の研究伴性継承2、突然変異率3、ニューラル システムの開発と細胞運命決定の4の重要な基礎知識にしかつながっていないが、また最近として浮上している、アルツハイマー病やパーキンソン病の5,6などいくつかの疾患に固有のメカニズムを研究する貴重なツールです。さらに、彼らは時間の短い期間で大量に発生する可能性が、寿命が短いと老化のプロセスを勉強する人気のあるモデルです。彼らはまた、インスリン産生細胞 (β 膵細胞に相当)、脂肪体 (同様には肝と白脂肪組織として機能している)、oenocytes (肝細胞様細胞)、心臓などの臓器に相同構造を所有しているだけでなく、体液代謝産物 (脊椎動物の血に似ています)7を輸送します。また、中間代謝 (インスリン/インスリン様成長因子のようなシグナル伝達経路およびラパマイシン ターゲット TOR 経路含む) の中央の道は、また非常に節約された7です。これらの理由から、ショウジョウバエは最近8糖尿病など代謝疾患固有の病態を中心に、代謝を制御する基本的なメカニズムを記述するため悪用されています。代謝の主要なコンポーネントは酸化的リン酸化プロセス (OXPHOS) を通じて ATP 生産、ミトコンドリア複数の経路を統合し、生命の最も重要な生物学的機能のいずれかを実行します。生物の代謝の中心的な役割を考慮したそれは、ミトコンドリアの機能障害がパーキンソン病9とアルツハイマー病10のような多くの病気だけでなく、筋萎縮性側索硬化症に関与していること意外ではないです。11,12. 老化プロセスの基本的な決定要因があります。確かに、酸化損傷11を通じて高濃度の細胞に有害なことができる細胞活性酸素種 (ROS) の主要な生産者しています。高齢化はまた破損または変異ミトコンドリア DNA13mitophagy 機能障害14,15ミトコンドリア生合成16障害の蓄積に関連付けられています。ミトコンドリアは細胞の恒常性の重要要因ではまた豊富や栄養素17,18の希少性によるといくつかの細胞機能を調節する別の基板を利用することができます。
確かに、ダイエット (炭水化物、脂質および蛋白質) のさまざまな栄養素に消化され、吸収され、細胞に運ばれます。細胞質に転化し、派生の基板は NADH や FADH219などの削減の同等物を製造しているミトコンドリアのマトリックスに運ばれます。これらの削減の同等は、電子輸送システム (ETS) の異なる酵素の複合体によって酸化されてし。これらの複合体は複合体など、ミトコンドリア内膜に埋め込まれている私と複合体 II。さらに、ミトコンドリアのグリセロール-3-リン酸脱水素酵素およびプロリン脱水素酵素など他の酵素の複合体は、ETS20,21に電子のエントリの代替ルートを表します。これらの 'alternative' 複合体は昆虫、特に重要として、種に応じて彼ら積極的に参加できる呼吸20,22,23,21を増加します。これらの ETS の供給システムからの電子は、最終的なアクセプター分子酸素まで、ユビキノン、その後複合体 III にし複雑な IV が転送されます。この電子の移動は、複雑な V (図 1) で ATP を ADP のリン酸化を運転内部のミトコンドリア膜プロトン動機力を生成します。病態生理学的ミトコンドリア細胞の恒常性は、キイロショウジョウバエ関連するモデルを表しますを用いたミトコンドリア代謝の勉強の中心的な役割の様々 なメカニズムを記述する強力なツールを検討条件または細胞と環境ストレスの下で。驚くほどしかし、研究の一握りだけは実際にショウジョウバエ24,25,26のミトコンドリアの呼吸を測定しました。確かに、ミトコンドリアの酸素消費量を評価することを目指して実験はミトコンドリアの分離を必要とします。分離が一般的にむしろ大量を必要とする (活性酸素産生やミトコンドリア効率27,28のマーカーとしての P/O 率) などさまざまなミトコンドリア機能の測定のために有利、いくつか個人24,29からのティッシュ。組織や個人の多量のためのこの要件は、重要な制限要因、特にすべての個人が同じ年齢であることを考えるとできれば実験のための同性の異なる時間に呼吸のメジャーを作る最高の状態で骨の折れるポイントします。さらに、ミトコンドリアの分離は、ミトコンドリア代謝を支配する根本的なメカニズムに重要な洞察力を提供できますが、ミトコンドリアの分離に使用するメソッドはレプリケート可能な結果を得ることの難しさなどいくつか欠点を持ってください。、ミトコンドリアのネットワークおよびミトコンドリアの構造と機能29,30,31の変質の中断。
本研究の目的は、非常に少数の個人から組織の最小限の量だけを用いたショウジョウバエのミトコンドリアの酸素消費量を測定するための堅牢なプロトコルを提示することです。このプロトコルでミトコンドリアの酸素消費量測定の in situショウジョウバエ thoraxes からグリセリン筋線維29を使用して高解像度呼吸32,33,との組み合わせで構成されています34,35ですこのメソッドも古典的なミトコンドリアの分離メソッドからセルの他のコンポーネントとの相互作用としても、ミトコンドリアの構造と機能はの保存よりグリセリンに比べて利点があります。繊維29,31,36, もっと生理学的に関連するこのアプローチになります。このプロトコルで、基板 ETS のいくつかの異なる段階で酸素消費量の決定をすること、ショウジョウバエの唯一の 3 つの thoraxes で使用する高解像度の呼吸ミトコンドリア機能することができます正確に評価されます。したがって、このプロトコルはショウジョウバエ モデルの利点を生かして多くの環境または病態生理学的条件のコンテキストで代謝を制御する基本的なメカニズムに関する質問に回答キーを助けることができます。
ETS のいくつかの異なる手順で酸素消費量を測定し、どのように異なる基板を評価する呼吸、別基板 (図 1)、ランプに貢献し、阻害剤が使用される30の透過後、組織。具体的には、異なる基板上の順次追加、ETS の異なる複合体を介して電子のエントリを刺激するために実行されます。シアン化物のカルボニル、ランプ 4-(フッ素系低分子) phenylhydrazone (FCCP) は非結合呼吸を測定するための最適な濃度で追加した、すなわち、非リン酸化呼吸筋酸素消費量刺激されます。複合体 I、II、および III は ETS 非酸化反応により、残留酸素の消費量を監視する実行し、順次抑制。N, N, N の注入による複雑な IV 最大呼吸能力を評価できる最後に、'、N - テトラメチル - p-フェニレンジアミン (受けた東京都警視庁)、人工電子プロバイダーとアスコルビン酸。それはハエの発生する温度なので、24 °C で実験が行われているに注意してくださいすることが重要です。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>High-resolution respirometer Oxygraph O2K</td>
			<td>Oroboros Instruments, Innsbruck, Austria</td>
			<td>10022-02</td>
			<td>Startup O2K respirometer kit 
			&#160;</td>
		</tr>
		<tr>
			<td>O2K-Titration Set&#160;</td>
			<td>Oroboros Instruments, Innsbruck, Austria</td>
			<td>20820-03</td>
			<td>Hamilton syringes with different volumes 
			&#160;</td>
		</tr>
		<tr>
			<td>Datlab software</td>
			<td>Oroboros Instruments, Innsbruck, Austria</td>
			<td>20700</td>
			<td>Software for data acquisition and analysis 
			&#160;</td>
		</tr>
		<tr>
			<td>Fine-tipped antimagnetic forceps</td>
			<td>VWR</td>
			<td>82027-400</td>
			<td> 
			&#160;</td>
		</tr>
		<tr>
			<td>Secura225D-1S-DQE</td>
			<td>Sartorius AG, Goettingen, Germany</td>
			<td></td>
			<td>Semi-micro balance (distributed by several companies)&#160; 
			&#160;</td>
		</tr>
		<tr>
			<td>Drosophila melanogaster wild-type w1118</td>
			<td>Bloomington Drosophila stock Center, IN, USA</td>
			<td></td>
			<td> 
			Storage Condition: 24 &#176;C</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>E4378</td>
			<td>EGTA 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Sigma-Aldrich</td>
			<td>P1767</td>
			<td>CAUTION: corrosive to metals, acute toxicity, skin corrosion, serious eye damage, acute aquatic toxicity. 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>CaCO3</td>
			<td>Sigma-Aldrich</td>
			<td>C4830</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>Na2ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A2383</td>
			<td> 
			Storage Condition: -20 &#176;C</td>
		</tr>
		<tr>
			<td>MgCl2.6H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M9272</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T0625</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>Na2Phosphocreatine</td>
			<td>Sigma-Aldrich</td>
			<td>P7936</td>
			<td> 
			Storage Condition: -20 &#176;C</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I5513</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td>D0632</td>
			<td> 
			Storage Condition: 2-8 &#176;C</td>
		</tr>
		<tr>
			<td>MES hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M8250</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>Saponin from quillaja bark</td>
			<td>Sigma-Aldrich</td>
			<td>S7900</td>
			<td>Saponin 
			Storage Condition: RT 
			Solution Preparation: 5 mg in 1 mL of preservation solution. Prepare fresh daily.</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P9791</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>05470</td>
			<td> 
			Storage Condition: 2-8 &#176;C</td>
		</tr>
		<tr>
			<td>Na2S2O4</td>
			<td>Sigma-Aldrich</td>
			<td>157953</td>
			<td>Sodium dithionite. CAUTION: self-heating substances and mixtures, acute toxicity, acute aquatic toxi chronic aquatic toxicity. 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P2256</td>
			<td>Pyruvate 
			Storage Condition: 2-8 &#176;C 
			Solution Preparation: In MilliQ water. Prepare fresh daily.</td>
		</tr>
		<tr>
			<td>L-(-)-Malic acid</td>
			<td>Sigma-Aldrich</td>
			<td>M1000</td>
			<td>Malate 
			Storage Condition: RT 
			Solution Preparation: In MilliQ water. Neutralize with KOH and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;-diphosphate monopotassium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A5285</td>
			<td>ADP 
			Storage Condition: -20 &#176;C 
			Solution Preparation: In MilliQ water. Neutralize with KOH and store at -80 &#176;C.</td>
		</tr>
		<tr>
			<td>Cytochrome c from equine heart</td>
			<td>Sigma-Aldrich</td>
			<td>C7752</td>
			<td>Cytochrome c 
			Storage Condition: -20 &#176;C 
			Solution Preparation: In MilliQ water. Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>L-Proline</td>
			<td>Sigma-Aldrich</td>
			<td>P0380</td>
			<td>Proline 
			Storage Condition: RT 
			Solution Preparation: In MilliQ water. Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Sodium succinate dibasic hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S2378</td>
			<td>Succinate 
			Storage Condition: RT 
			Solution Preparation: In MilliQ water. Neutralize with HCl and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>sn-Glycerol 3-phosphate bis(cyclohexylammonium) salt</td>
			<td>Sigma-Aldrich</td>
			<td>G7886</td>
			<td>Glycerol-3-phosphate 
			Storage Condition: -20 &#176;C 
			Solution Preparation: In MilliQ water. Neutralize with HCl and store at -80 &#176;C.</td>
		</tr>
		<tr>
			<td>Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone</td>
			<td>Sigma-Aldrich</td>
			<td>C2920</td>
			<td>FCCP. CAUTION: acute toxicity, skin sensitisation, chronic aquatic toxicity. 
			Storage Condition: RT 
			Solution Preparation: In absolute ethanol. Store in glass vials at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875</td>
			<td>CAUTION: acute toxicity, skin irritation, eye irritation, specific target organ toxicity (respir sytem), acute aquatic toxicity, chronic aquatic toxicity. 
			Solution Preparation: In absolute ethanol. Store in dark vials at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Malonic acid</td>
			<td>Sigma-Aldrich</td>
			<td>M1296</td>
			<td>Malonate. CAUTION: acute toxicity, serious eye damage. 
			Storage Condition: RT 
			Solution Preparation: In MilliQ water. Neutralize with KOH. Prepare fresh daily.</td>
		</tr>
		<tr>
			<td>Antimycin A from Streptomyces sp.</td>
			<td>Sigma-Aldrich</td>
			<td>A8674</td>
			<td>Antimycin A. CAUTION: acute toxicity, acute aquatic toxicity, chronic aquatic toxicity. 
			Storage Condition: -20 &#176;C 
			Solution Preparation: In absolute ethanol. Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>N,N,N&#8242;,N&#8242;-Tetramethyl-p-phenylenediamine</td>
			<td>Sigma-Aldrich</td>
			<td>T7394</td>
			<td>TMPD 
			Storage Condition: RT 
			Solution Preparation: In MilliQ water. Store in dark vials at&#160; -20 &#176;C.</td>
		</tr>
		<tr>
			<td>(+)-Sodium L-ascorbate</td>
			<td>Sigma-Aldrich</td>
			<td>A4034</td>
			<td>Ascorbate 
			Storage Condition: RT 
			Solution Preparation: In MilliQ water. Store in dark vials at&#160; -20 &#176;C.</td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td>Sodium azide. CAUTION: acute toxicity (oral and dermal), specific target organ toxicity (brain), aquatic toxicity, chronic aquatic toxicity.&#160; 
			Solution Preparation: In MilliQ water. Store at -20 &#176;C.</td>
		</tr>
	</tbody>
</table>

measurement of mitochondrial oxygen consumption in permeabilized fibers of drosophila using minimal amounts of tissue

ショウジョウバエ、キイロショウジョウバエ代謝の研究のための新たなモデルを表します。確かに、ショウジョウバエは、構造の人間の臓器に相同性を持つし、非常に節約された代謝経路を持っている時間の短い期間で別の基礎的メカニズムの研究を可能にする比較的短い寿命を持っています。しかし、意外な細胞の代謝、ミトコンドリア呼吸に不可欠なメカニズムの 1 つは徹底的に触れられていないこのモデルです。ショウジョウバエにおけるミトコンドリア呼吸の測定は通常非常に多数の個人を必要とし、得られた結果が再現性が高いのでそうです。ここでは、ショウジョウバエから組織の最小限の量を使用してミトコンドリアの酸素消費量の正確な測定を許可する方法を説明します。この方法では、thoraxes を切除し、両方鋭い鉗子で機械的と化学的にサポニン、異なる化合物は細胞膜を通過し、ミトコンドリア呼吸を調節することができますグリセリンです。透過処理、したプロトコルが、ランプといくつかの阻害剤に彼らの対応だけでなく、異なる基質を酸化する電子輸送システム (ETS) の異なる複合体のキャパシティを評価する実行されます。このメソッドは、ミトコンドリアがまだ他の細胞成分との相互作用とミトコンドリアの形態が保存されているので、それはもっと生理学的に関連するとミトコンドリアの分離を用いた方法と比較して多くの利点を示します。また、サンプル準備の速度が速い、得られた結果が再現性が高い。ミトコンドリア呼吸性と代謝の研究のためのモデルとしてショウジョウバエの利点を組み合わせることにより新たな洞察が、発表することができます重要な特にときハエが発生している異なる環境や病態生理学的条件。

上記で説明したプロトコルを使用してミトコンドリアの酸素消費量の代表的なトレースは、図 2で提供しています。ピルビン酸とリンゴ酸グリセリン筋線維とともにチャンバーに注入、呼ばれる CI リーク呼吸、すなわち、複雑な ETS の私がによって刺激されるとき、NADH 生産にピルビン酸とリンゴ酸による酸化を介して、トリカルボン酸 (C...

Watch this Scientific Journal Video about Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue at JoVE.com

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

本稿で、ショウジョウバエのグリセリン thoraxes による高解像度の呼吸の酸素消費量を測定する方法を説明します。この手法は古典的なミトコンドリアの分離法と比較して組織の最小限の量を必要として得られた結果、もっと生理学的に関連します。

ショウジョウバエのティッシュの最小限の量を使用してのグリセリンの繊維でミトコンドリアの酸素消費量の測定

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

measurement-mitochondrial-oxygen-consumption-permeabilized-fibers

Research

JoVE Journal

Biochemistry

11.8K ビュー.  Université de Moncton. 本稿で、ショウジョウバエのグリセリン thoraxes による高解像度の呼吸の酸素消費量を測定する方法を説明します。この手法は古典的なミトコンドリアの分離法と比較して組織の最小限の量を必要として得られた結果、もっと生理学的に関連します。

ビデオ: Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

足場リポソームを使用することにより、脂質近位タンパク質 - タンパク質相互作用を再構成します<em&gt;インビトロ</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

生化学

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include flavin-dependent respiratory complexes and dehydrogenases that produce a mixture of superoxide (O2&#9679;-) and hydrogen peroxide (H2O2). Accurate quantification of the ROS-producing potential of individual sites in isolated mitochondria can be challenging due to the presence of antioxidant defense systems&#160;and side reactions that also form O2&#9679;-/H2O2. Use&#160;of nonspecific inhibitors that can disrupt mitochondrial bioenergetics can also compromise measurements by altering&#160;ROS release from other sites of production. Here, we present an easy method for the simultaneous measurement of H2O2 release and nicotinamide adenine dinucleotide (NADH) production by purified flavin-linked dehydrogenases. For our purposes here, we have used purified pyruvate dehydrogenase complex (PDHC) and &#945;-ketoglutarate dehydrogenase complex (KGDHC) of porcine heart origin as examples. This method allows for an accurate measure of native H2O2 release rates by individual sites of production by eliminating other potential sources of ROS and antioxidant systems. In addition, this method allows for a direct comparison of the relationship between H2O2 release and enzyme activity and the screening of the effectiveness and selectivity of inhibitors for ROS production. Overall, this approach can allow for the in-depth assessment of native rates of ROS release for individual enzymes prior to conducting more sophisticated experiments with isolated mitochondria or permeabilized muscle fiber.

Mitochondria contain several flavin-dependent enzymes that can produce reactive oxygen species (ROS). Monitoring ROS release from individual sites in mitochondria is challenging due to unwanted side reactions. We present an easy, inexpensive method for direct assessment of native rates for ROS release using purified flavoenzymes and microplate fluorometry.

活性酸素・過酸化水素とフラビン含有ミトコンドリア脱水素酵素による NADH 生産の同時測定

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

ミトコンドリアの分離と培養細胞におけるミトコンドリアのカルシウム流入の解析

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert NADH and FADH into ATP. Since mitochondria are also the primary site for oxygen use and nutrient oxidation to carbon dioxide and water, how efficiently they use oxygen and produce ATP directly relates to the efficiency of nutrient metabolism, nutrient requirements of the animal, and health of the animal. The purpose of this method is to examine mitochondrial respiration, which can be used to examine the effects of different drugs, diets and environmental effects on mitochondrial metabolism. Results include oxygen consumption measured as proton dependent respiration (State 3) and proton leak dependent respiration (State 4). The ratio of State 3 / State 4 respiration is defined as respiratory control ratio (RCR) and can represent mitochondrial energetic efficiency. Mitochondrial proton leak is a process that allows dissipation of mitochondrial membrane potential (MMP) by uncoupling oxidative phosphorylation from ADP decreasing the efficiency of ATP synthesis. Oxygen and TRMP+ sensitive electrodes with mitochondrial substrates and electron transport chain inhibitors are used to measure State 3 and State 4 respiration, mitochondrial membrane PMF (or the potential to produce ATP) and proton leak. Limitations to this method are that liver tissue must be as fresh as possible and all biopsies and assays must be performed in less than 10 h. This limits the number of samples that can be collected and processed by a single person in a day to approximately 5. However, only 1 g of liver tissue is needed, so in large animals, such as dairy cattle, the amount of sample needed is small relative to liver size and there is little recovery time needed.

Here, we share methods for measuring mitochondrial oxygen consumption, a defining concept of nutritional energetics, and proton leak, the primary cause of inefficiency in mitochondrial generation of ATP. These results can account for 30% of the energy lost in nutrient utilization to help evaluate mitochondrial function.

ホルスタイン種乳牛におけるミトコンドリア呼吸を推定する肝ミトコンドリアの酸素消費量とプロトン リーク動態測定

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

測定と酸素消費量全体の解釈飛ぶ頭セグメント

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Direct Measurement of KDM1A Target Engagement Using Chemoprobe-based Immunoassays

The assessment of the target engagement, defined as the interaction of a drug with the protein it was designed for, is a basic requirement for the interpretation of the biological activity of any compound in drug development or in basic research projects. In epigenetics, target engagement is most often assessed by the analysis of proxy markers instead of measuring the union of the compound to the target. Downstream biological readouts that have been analyzed include the histone mark modulation or gene expression changes. KDM1A is a lysine demethylase that removes methyl groups from mono- and dimethylated H3K4, a modification associated with the silencing of gene expression. Modulation of the proxy markers is dependent on the cell type and function of the genetic make-up of the cells investigated, which can make interpretation and cross-case comparison quite difficult. To circumvent these problems, a versatile protocol is presented to assess the dose effects and dynamics of the direct KDM1A target engagement. The assay described makes use of a KDM1A chemoprobe to capture and quantify uninhibited enzyme, can be broadly applied to cells or tissue samples without the need for genetic modification, has an excellent window of detection, and can be used both for basic research and analysis of clinical samples.

Here, we present a protocol to measure KDM1A target engagement in a human or animal cell, tissue or blood samples treated with KDM1A inhibitors. The protocol employs chemoprobe tagging of the free KDM1A enzyme and direct quantification of the target occupation using chemoprobe-based immunoassays and can be used in preclinical and clinical studies.

ケモプローブベースの免疫アッセイを用いてKDM1A標的エンゲージメントを直接測定

The assessment of the target engagement, defined as the interaction of a drug with the protein it was designed for, is a basic requirement for the ...

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective denaturation of proteins from RNA, 3) ribonuclease inactivation, and 4) removal of contamination from DNA, proteins, and carbohydrates. However, it is very laborious to maintain the integrity of RNA when there are high levels of RNase in the tissue. Spontaneous autolysis makes it very difficult to extract RNA from pancreatic tissue without damaging it. Thus, a practical RNA extraction method is needed to maintain the integrity of pancreatic tissues during the extraction process. An experimental and comparative study of existing protocols was carried out by obtaining 20-30 mg of rat pancreatic tissues in less than 2 minutes and extracting the RNA. The results were assessed by electrophoresis. The experiments were carried out three times for generalization of the results. Immersing pancreatic tissue in RNA stabilization reagent at -80 &#176;C for 24 h yielded high integrity RNA, when the RNA extraction reagent was used as the reagent. The results obtained were comparable to the results obtained from commercial kits with spin column bindings.

The purity and integrity of the isolated RNA is a vital step in RNA dependent assays. Here, we present a practical, rapid, and inexpensive method to extract RNA from a small quantity of undamaged pancreatic tissue.

ラット膵臓組織の迅速かつ費用対効果の高いRNA抽出

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.

Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

ソウの子孫から以前に凍結した心臓組織におけるミトコンドリア電子伝達複合体の測定:運動誘発ミトコンドリアバイオエネルギーの変化を評価するモデル

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis ...

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.

A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

透過型電子顕微鏡による骨格筋線維の細胞内グリコーゲン分布の定量化

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

<meta charset="utf8"></head><body>3Dプリントされたスタンプ状デバイスによる組織スフェロイドの生成

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

3Dプリントされたスタンプ状デバイス による 組織スフェロイドの生成

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...