我々は、ミトコンドリアの生物学の分野への著者の紹介と、この手順では多くの有用な洞察とアドバイスを教授A.ビノグラードフ博士とヴェラGrivennikovaに感謝。著者らは、ビデオの準備のヘルプはクイーンズ大学メディアオフィスからミセスアマンダMcKintockに感謝しています。

はじめラットの心臓からミトコンドリアは細胞代謝の過去と現在の研究のための最も一般的な準備の一つです。彼らは、野生型またはノックアウト動物から大量に迅速かつ確実に得ることができます。一般的な手順は、トリプシン、分画および分画遠心法によって組織の消化で​​構成されています。 心臓（図1）を含む、様々な組織からミトコンドリアの単離中に保持しなければならないいくつかの条件があります。 <ul><li>組織は、それが直接得られるミトコンドリアの収量に影響を及ぼすので徹底的に、みじん切りする必要があります。心臓組織は、小さな曲線ハサミでミンチに最適ですし、組織グラインダーは、1.0前後直径の出口孔径とガーリックプレス利用できない場合は、Latapie組織グラインダーを使用するかを続けることができます。 </li><li>それは、各ステップでの解の温度を維持するために必要です。したがって、全体の手順は、寒い部屋とすべての機器で行う​​必要があるとソリューションは、事前に準備して冷却する必要があります。温度条件が保存されていない場合は準備の安定的ないくつかのプロパティが失われることがあります。ミトコンドリアの構造は、能動輸送の研究が重要な場合は特に、凍結ので、懸濁液のoverfreezing（遠心分離時など）に非常に敏感であることは許可されていません。 </li><li>重要なパラメータは、プロシージャの時間の長さであり、分離は、可能な限り迅速に実施して得られる製剤は直ちに使用する必要がありますする必要があります。したがって、手術器具、ソリューション及びアプライアンスは、事前に準備する必要があります。 </li></ul>材料と器具楽器<ol><li>ストレートオペレーティングはさみ、鋭いポイント</li><li>湾曲したハサミ</li><li>鉗子</li><li>ペトリ皿</li><li>小さな漏斗+ナイロンフィルターの一部（洗ってある） </li><li> 6ビーカー50ミリリットル</li><li>二つのマグネットスターラー二氷浴</li><li>一つの大きな氷浴</li><li> 2つの小さな磁石のバー</li><li>掻きペレット用ヘラ</li><li>遠心チューブ</li><li> Latapie組織のプレス（ガーリックプレスで置換することができます） </li><li> 20ミリリットルと1〜2ミリリットルテフロン - ガラスのhomogenisers </li><li>廃棄物のビーカー</li><li>タンパク質測定のための最終的なミトコンドリア懸濁液と試料のエッペンドルフチュー​​ブ</li><li>氷浴</li></ol>ソリューション（2匹ごとに計算）： <ol><li>洗浄緩衝液：0.3 Mのショ糖、10mMのHEPES（pH値= 7.2）、0.2mMのEDTA（1000 ml）を</li><li>絶縁バッファ：0.3 Mのショ糖、10mMのHEPES（pH = 7.4）で、0.2mMのEDTA、1 mg / mlのBSA（シグマA6003）（100ミリリットルの洗浄用バッファーから調製）。ウシ血清アルブミンは、より安価な脂肪酸を含まない類縁体で置換することができる。同じバッファまたはその等張バリエーションがバッファを再懸として使用することができます。 </li><li>トリプシン（シグマT8003）解決策：1mMのHCl中で2.5mg/ml（0.5 ml）を</li><li>大豆（シグマT9128）絶縁バッファの6.5 mg/10 mlのトリプシンインヒビター</li></ol>プロトコル手順の一般的なスキームを図に示されています。 1。心臓を冷却し、バッファ、心室組織は、摘出したみじん切りとトリプシンとの光分解治療中に均質洗濯で洗浄する必要があります。懸濁液を低速で遠心分離し、得られた上清は、ミトコンドリアを収集するためにより高い速度で再び遠心分離する。計画された実験に応じて再懸濁バッファーは、他の等張液で置き換えることができます。 動物は、英国に基づき終了された&quot;動物（科学的手続）法。&quot;頸椎脱臼によって斬首によって続く。それは、動物の終端と心臓の抽出の間に遅延がないように、すぐに動物の斬首後に心臓を取り出すようにしなければなりません。肝ミトコンドリアのパラレル分離が期待されている場合、ラットは前の実験に一晩絶食されるべきである。 <ol><li>洗浄バッファー40mlを備えた三ビーカーを準備します。次の手順に進む前に3〜5分間、氷塩浴でそれらを冷却する。それらをover​​freezeと氷の結晶の雲が現われ始める直後にすぐに使用しないことを確認してください。 </li><li>首を切らラットの胸部を開き、心を抽出する。小さな切開を行い、その後すぐに最初のビーカーに氷冷溶液に心を移す。血液を除去すると第二のビーカーにそれを置くために心を絞る。それぞれの心のためにこの操作を繰り返します。 </li><li>ろ紙上に心を乾かします。すべての脂肪、血餅、耳介や筋膜やプール心室組織を削除します。 </li><li>粒子の大きさは1-2 mm程度になるまで約5分間氷上でペトリ皿上で小さな曲面ハサミでプールされた組織をミンチ。 </li><li>組織のプレスとパスにみじん切りの組織を置くそれを通して。材料のかなりの量が非常にマスコミの壁や底部からすべての心臓組織を収集する記者に残ることがあるので注意してください。 </li><li> 50 mlの洗浄バッファーをビーカーに材料を移し、かき混ぜる。その後、ナイロンフィルタを介してサスペンションにフィルタをかける。洗浄バッファーでフィルターを3回にみじん切りの組織を洗浄し、ろ液を捨てる。 </li><li>洗浄緩衝液20ml中に、洗浄した組織を移し、マグネチックスターラー上に氷浴に入れてください。一定速度で撹拌下でトリプシン溶液0.5mlを追加し、タイマーを起動します。別のマグネチックスターラー、氷浴と第50 mlのビーカーを用意します。 </li><li> 第 4 回分で簡単にサスペンションを分散させるために最大で数とダウンストロークで小さな遊嵌ガラス-テフロンホモジナイザーを（10〜15ミリリットル）を使用して部分によってサスペンションの部分を均質。均質化した後に第二のビーカーに懸濁液を移す。あなたが全体の中の懸濁液の均質化を2回行うように、 第 9 回分でこの手順を繰り返します。インキュベーションの15分後、バッファを含むトリプシンインヒビターを分離10mlを加え、1分間インキュベートする。 </li><li>ナイロンフィルターに通し、再び懸濁物を濾過し、ろ液を保存する。洗浄バッファーの15〜20ミリリットルで1分間フィルタと均質に残された微粒子画分を集める。 </li><li>ろ液とのホモジネートを組み合わせると600グラムで15分間遠心操作。 </li><li>慎重に新しい遠心管に得られた上清をフィルターします。ペレットによる汚染を回避し、8500グラムで15分間再遠心する。 </li><li>高速遠心分離後上清を破棄し、バッファを破棄白色綿毛状の外縁層1mlでペレット2〜3回すすいでください。スパチュラでペレットを破るが一番下にある赤血球の赤色スポットを避ける。血液細胞はロースを取得した場合、均質化する前に懸濁液から赤のビットを削除する。 </li><li>穏やかなピペッティングによる小ホモジナイザーあるいはでペレットを再懸濁します。より多くのIsolationbufferで希薄懸濁液と8500グラムで15分間再遠心する。 </li><li>上清を廃棄し、バッファを（任意の等張緩衝液）再懸でペレットを再懸濁し、再び遠心する。 </li><li>得られた茶色のペレットは、洗浄後そのままミトコンドリアが含まれています。お好みの等張緩衝の非常に小さな音量でペレットを再懸濁する。 </li></ol><img src="/files/ftp_upload/2202/2202fig1.jpg" alt="図1" /> ミトコンドリアラットの心臓の分離のステップの手順でステップを示す図1。スキーム。上部、ステージ1-9：組織破壊、トリプシン処理し、均質化、底の部分、ステージ10-15：分画遠心法。

<ol>
	<li>Jacobus, W. E., Saks, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creatine+kinase+of+heart+mitochondria:+changes+in+its+kinetic+properties+induced+by+coupling+to+oxidative+phosphorylation.">Creatine kinase of heart mitochondria: changes in its kinetic properties induced by coupling to oxidative phosphorylation.</a> Arch. Biochem. Biophys. 219, 167-178 (1982).</li><li>Gostimskaya, I. S., Grivennikova, V. G., Zharova, T. V., Bakeeva, L. E., Vinogradov, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+assay+of+the+intramitochondrial+enzymes:+use+of+alamethicin+for+permeabilization+of+mitochondria.">In situ assay of the intramitochondrial enzymes: use of alamethicin for permeabilization of mitochondria.</a> Anal. Biochem. 313, 46-52 (2003).</li></ol>

利害の衝突は宣言されません。

結合度の高い無傷ミトコンドリアの単離のための詳細な迅速なプロトコルが記述されています。て得られる製剤は、細胞生体エネルギー、プロテオミクス研究やミトコンドリアDNAと脂質含量の分析に機能的または構造的な研究に使用することができます。イオンと代謝中間体の能動輸送の生物物理学的研究も行うことができます。さらに亜ミトコンドリアの粒子、外膜を分離したり、酸化的リン酸化の別個の成分を浄化するために、ミトコンドリアの準備を処理することが可能です。記載された方法はジャコバス、サックス1による分離の標準的なプロトコルの変更です。準備ミトコンドリアの予想収量は、心臓あたりのミトコンドリアタンパク質の約10〜15ミリグラムであり、そのような準備のリンゴ酸/グルタミン酸に対する呼吸調節率は0.12前後モルO 2 xminは-1 XMGのタンパク質の状態IVの呼吸、8〜12の間に変化する23 -1 ° 0.25 Mショ糖を含む培地中でC、10mMの塩化カリウム、0.2mMのEDTA、正確な実験条件および付加のためのイニシエーションの状態IIIの呼吸（150μMADPで5 mMリン酸カリウム（pH8.0）に、refを参照してください2）。 特別な注意が分離手続の開始前にいくつかの技術的な詳細に与えられるべきである。それは、チューブが完全にクリーンなブラシや温水で洗浄する必要があるため、クリーンなポリカーボネートやポリプロピレン遠心チューブを使用することが重要ですが、界面活性剤処理は必要最小限度のものとすべきである。また、それは分離前日寒い部屋に必要なすべてのガラス器具や楽器を収集することをお勧めします。 隔離中にフラスコからビーカーにバッファを転送する際に、а特別な注意は、ソリューションに氷水の低下を防ぐために与えられるべきである。組織を取り扱う際には、心臓の抽出は、動物の斬首と組織を冷却する間にも短い時間遅延は部分的に非結合ミトコンドリアの結果になるので、できるだけ早く実施すべきであることを強調しておきたい。急速冷却と削除された心の徹底的な洗浄は、ヘモグロビンと赤血球NADaseからフリー、標準製剤を得ることが不可欠である。 トリプシンは、均質化中に、せん断力に組織の感受性を高めるために結合するタンパク質の分解に使用されます。それは低品質のミトコンドリアにつながるので、プロテアーゼの組織の長期ばく露は避けるべきである。 高速遠心後の遠心チューブの壁は、壁に蓄積された脂肪物質の付着物を除去するためにティッシュペーパーで拭き取ってください。 最終的な再懸濁するためには、貯蔵中のミト​​コンドリアの機能（心臓あたりのバッファ150〜200μlの）の損失を最小限に抑えるために、最終的なバッファの低容量を使用することをお勧めします。二価陽イオンの輸送の研究にはキレート剤は、EGTAを含まない培地で数回洗浄し、分離後の最初の時間内で使用されるべきであるため、ミトコンドリア、最終調製物中に存在してはならない。

preparation of highly coupled rat heart mitochondria

酸化的リン酸化の過程で細胞内のATPの世代にミトコンドリアの機能が広く認識されている。過去数十年の間にエネルギーの世代よりもミトコンドリアの他の機能の理解に大きな進歩がありました。これらは、シグナル伝達細胞小器官、哺乳類の開発と高齢化だけでなく、細胞の代謝と細胞増殖の間の調整への貢献として機能する、アポトーシスにおける役割が含まれています。ミトコンドリアで変調された生物学的プロセスの理解が分離し、実験動物の組織から無傷のミトコンドリアの処理のための堅牢な方法に基づいています。ラットの心臓からミトコンドリアは、ノックアウト動物に関する研究を含む細胞代謝の過去と現在の研究のための最も一般的な準備の一つです。 ここでは、結合度の高い無傷ミトコンドリアの単離のための詳細な迅速な方法を説明します。ミトコンドリアラットの心臓のような製剤は、細胞の生体エネルギー、生体分子の輸送、プロテオミクス研究やミトコンドリアDNA、タンパク質や脂質の分析上の機能と構造の研究のための優れたオブジェクトです。

Watch this Scientific Journal Video about Preparation of Highly Coupled Rat Heart Mitochondria at JoVE.com

Preparation of Highly Coupled Rat Heart Mitochondria

我々は細胞の生体エネルギー、生物物理学的測定、プロテオミクスやミトコンドリアDNAと脂質の分析の機能的または構造的研究のためのミトコンドリア純粋な、高度に結合したラット心臓の分離のためのаのプロトコルについて説明します。

ミトコンドリアは密結合のラットの心臓の準備

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

preparation-of-highly-coupled-rat-heart-mitochondria

Research

JoVE Journal

Biology

22.7K ビュー.  University of Manchester. 我々は細胞の生体エネルギー、生物物理学的測定、プロテオミクスやミトコンドリアDNAと脂質の分析の機能的または構造的研究のためのミトコンドリア純粋な、高度に結合したラット心臓の分離のためのаのプロトコルについて説明します。

ビデオ: Preparation of Highly Coupled Rat Heart Mitochondria

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

ラット心臓における培養組織の移植

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

生物学

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

成体ラットの心筋細胞の初代培養

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

酵母細胞からミトコンドリアの精製

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

の電子顕微鏡のための胚の調製ショウジョウバエ胎児心臓の管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

生体力学とMechanobiological研究ラット尾腱の準備

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

単離ミトコンドリアマウス心臓における透水性遷移孔の開口部のマルチパラメータ測定

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

シングルミトコンドリアスーパーオキシド無傷ハートや点滅の共焦点イメージング<em&gt;インビボ</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

哺乳類の全胚培養に適したラット血清の調製

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

自動化されたモジュラーハイスループットエキソポリサッカライドスクリーニングプラットフォームは、高感度炭水化物フィンガープリント分析と相まって

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

単離されたワーキングラット心臓におけるグルコースおよび脂肪酸酸化の宿泊料金を決意するための方法

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...