この作品は、CIHRとカナダのNSERCからの補助金によって支えられている。 VITはCIHR新調査官とゲノミクスのコンコルディア大学のリサーチチェア、細胞生物学と高齢化です。

材料および方法酵母菌株及び成長条件野生型株BY4742（MATαhis3Δ1leu2Δ0lys2Δ0ura3Δ0が ）豊富なYEPD培地（1％酵母エキス、2％ペプトン、2％グルコース）で増殖させた。細胞を30℃で回転が5:1の&quot;フラスコの体積/培地体積&quot;比での三角フラスコで200 rpmで振盪しながら培養した。 原油ミトコンドリア分画の単離<ol><li> 48時間野生型株BY4742培養液1 Lに成長する</li><li> 500mlのNalgeneの遠心管に文化を注ぐ。室温で3000 × gで5分間の遠心分離により、負荷や収穫の細胞のバランスをとる。 </li><li> dH2Oの250mlの上清をデカントし、ペレット化した細胞懸濁する。室温で3000 × gで5分間の遠心分離により細胞をペレット化する。 </li><li> dH2Oの250mlの上清をデカントし、ペレット化した細胞懸濁する。室温で3000 × gで5分間の遠心分離により細胞をペレット化する。 </li><li>細胞ペレットの湿重量を決定します。 </li><li> DTTバッファーでペレット化した細胞を再懸[バッファー/ g（湿重量）の細胞を2ml]。 </li><li> 50 mlのファルコンのプラスチックチューブに細胞懸濁液を移す。 </li><li> 30℃で20分間シェーカーで70 rpmでチューブを回転させる</li><li>室温で3000 × gで5分間の遠心分離により細胞を回収。 </li><li>ザイモリアーゼ[バッファ/ g（湿重量）の細胞7mlの] なしザイモリアーゼバッファーでペレット化した細胞を再懸濁します。 </li><li>室温で3000 × gで5分間の遠心分離により細胞をペレット化する。 </li><li>ザイモリアーゼ[バッファ/ g（湿重量）の細胞7mlの] なしザイモリアーゼバッファーでペレット化した細胞を再懸濁します。 </li><li>ガラス製フラスコに細胞懸濁液を移す。 </li><li>ザイモリアーゼ- 100T [ザイモリアーゼ- 100T / g（湿重量）の細胞を1mg]細胞懸濁液への粉体を​​追加。 </li><li> 30℃で30分間シェーカーで70 rpmで細胞懸濁液でフラスコを回転させる</li><li>転送フェロプラストは、ザイモリアーゼ- 100T〜50 mlのプラスチック遠心管で細胞壁の消化のために結成。 </li><li> 4℃で2200 × gで8分間℃での遠心分離によりペレットのスフェロプラスト</li></ol> **すべての後続のステップは4℃または氷上で行ってください。スフェロプラストの懸濁液を穏やかに細胞小器官を破ることを避けるため、カットのヒントをピペットを用いて処理する必要があります** <ol start="18"><li>氷冷均質化緩衝液[バッファ/ g（湿重量）の細胞の6.5 ML]でペレットスフェロプラストを再懸濁します。 </li><li> 4℃で2200 × gで8分間℃での遠心分離によりペレットのスフェロプラスト</li><li>氷冷均質化緩衝液[バッファ/ g（湿重量）の細胞の6.5 ML]でペレットスフェロプラストを再懸濁します。 </li><li>氷ガラスホモジナイザーで予備冷却するセルを転送する。 </li><li>タイトな乳棒を用いて、乳棒の15ストロークをすることで細胞をホモジナイズする。 </li><li>氷冷均質化緩衝液の1ボリュームを追加します。 </li><li> 50 mlのプラスチック遠心管に均質化スフェロプラストを転送する。 </li><li> 4℃で1500 × gで5分間遠心してペレット切れ目のない細胞、核、および大規模な破片℃の</li><li> 4℃で3000 × gで5分間得られた上清を遠心℃に</li><li> 4℃をXGの12000で15分間、得られた上清を遠心</li><li>氷冷均質化緩衝液の上清をデカントし、ペレットを再懸濁します[バッファ/ g（湿重量）の細胞の6.5ミリリットル]。 </li><li> 4℃で3000 × gで5分間遠心する℃、 </li><li> 4℃をXGの12000で15分間、得られた上清を遠心</li><li>氷冷SEMの緩衝液3mlで上清をデカントし、ペレットを再懸濁します。 </li></ol> **この懸濁液をミトコンドリアに富んでいるものの、それはまた、小胞体（ミクロソーム）、ゴルジ体、および液胞などの他の小器官が含まれています。純粋なミトコンドリアを取得するには、この粗ミトコンドリア分画は、後述のように、さらに分別にかけることができます** 他の小器官による汚染のミトコンドリアを欠いての精製<ol><li> EMの氷冷60％（w / v）ショ糖の代わりに1.5ミリリットルのベックマンウルトラクリア遠心管にバッファリング。 </li><li>オーバーレイ60％（W / V）32％、23％液1.5 ml、15％スクロース（EMバッファ内のすべての重量/ v）を1.5 mlの4 mlのショ糖。 </li><li> 15％（w / v）スクロースの上にSEMバッファの粗ミトコンドリア分画の3mlを置きます。 </li><li> 4で134000 XG（33000回転）℃で1時間のためのベックマンSW41のTiスイングローターで遠心</li><li> 60％/ 32％スクロース界面で無傷のミトコンドリア形態は茶色のバンド。 </li><li>慎重にミトコンドリアのバンドに到達するまで、ショ糖を削除します。 </li><li>カットの先端にピペットを用いてミトコンドリアのバンドを外して、MLS - 5ローターのためベックマン遠心管に入れてください。 </li><li> SEM用緩衝液でチューブを埋める。 </li><li>ペレット4℃で30分10000 × gで（31000 rpm）で30分間MLS - 5ローターの遠心分離によって純粋なミトコンドリア℃の</li><li>使用して上清をデカントピペット。 </li><li>ミトコンドリア機能、ミトコンドリアプロテオームとlipidome、およびミトコンドリアDNAの分析のための純粋なミトコンドリアを使用してください。 </li></ol>試薬<ol><li> DTTバッファー[100 mMのTris/H2SO4液（pH 9.4）、10mMのジチオスレイトール]（15ミリリットル用） <ul style="list-style-type:none;"><li> 1 M Tris/H2SO4緩衝液（pH 9.4）1.5 mlの</li><li> 1 M DTTを150μl </li><li> 15mlに容積</li></ul></li><li>ザイモリアーゼバッファー[20 mMリン酸カリウム（pH7.4）に、1.2 Mソルビトール]（100ミリリットル用） <ul style="list-style-type:none;"><li> 1 Mリン酸カリウム緩衝液（pH7.4）を2ml </li><li>ソルビトール2 M 60 mlの</li><li> 100mlにボリューム</li></ul></li><li>均質化緩衝液[10mMトリス/ HCl（pH7.4）中、ソルビトール0.6 M、1mMのEDTA、0.2％（w / v）のBSA]（250ミリリットル用] <ul style="list-style-type:none;"><li> 1 Mトリス/ HCl緩衝液（pH7.4）2.5 mlの</li><li>ソルビトール2 M 75mlの</li><li> 500mMのEDTAを500μl </li><li> BSA、0.5gの</li><li> 250mlにボリューム</li></ul></li><li> SEMバッファー[10 mMのMOPS / KOH（pH7.2）に、250mMスクロース、1mMのEDTA]（250ミリリットル用） <ul style="list-style-type:none;"><li> 1 MのMOPS / KOH緩衝液（pH7.2）2.5 mlの</li><li> 2 Mのショ糖の31.25ミリリットル</li><li> 500mMのEDTAを500μl </li><li> 250mlにボリューム</li></ul></li><li> EMバッファー[10mMのMOPS / KOH（pH7.2）中、1mMのEDTA]（250ミリリットル用） <ul style="list-style-type:none;"><li> 1 MのMOPS / KOH緩衝液（pH7.2）2.5 mlの</li><li> 500mMのEDTAを500μl </li><li> 250mlにボリューム</li></ul></li></ol>

<ol>
	<li>Voet, D., Voet, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry.">Biochemistry.</a> , (2004).</li><li>Suen, D. -. F., Norris, K. L., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dynamics+and+apoptosis.">Mitochondrial dynamics and apoptosis.</a> Genes Dev. 22, 1577-1590 (2008).</li><li>Cheng, W. C., Berman, S. B., Ivanovska, I., Jonas, E. A., Lee, S. J., Chen, Y., Kaczmarek, L. K., Pineda, F. &. a. m. p. ;. a. m. p., Hardwick, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+factors+with+dual+roles+in+death+and+survival.">Mitochondrial factors with dual roles in death and survival.</a> Oncogene. 25, 4697-4705 (2006).</li><li>Detmer, S. A., Chan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+and+dysfunctions+of+mitochondrial+dynamics.">Functions and dysfunctions of mitochondrial dynamics.</a> Nat. Rev. Mol. Cell Biol. 8, 870-879 (2007).</li><li>McBride, H. M., Neuspiel, M., Wasiak, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+more+than+just+a+powerhouse.">Mitochondria: more than just a powerhouse.</a> Curr. Biol. 16, R551-R560 (2006).</li><li>Balaban, R. S., Nemoto, S., Finkel, 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,+oxidants,+and+aging.">Mitochondria, oxidants, and aging.</a> Cell. 120, 483-495 (2005).</li></ol>

The authors have nothing to disclose.

このメソッドは、酵母細胞から純粋なミトコンドリアの高収量を可能にします。この方法で精製したミトコンドリアは、他の小器官で、本質的に汚染の自由であり、その精製後、構造と機能の整合性を保持。本質的なミトコンドリアのプロセスの無細胞再構成に適しているミトコンドリアに記載の方法利回り。これらの高純度のミトコンドリアは、ミトコンドリアの構造と機能、ミトコンド?...

purification of mitochondria from yeast cells

ミトコンドリアは、真核生物、非光合成細胞における好気的代謝の間にATPの生産の主なサイトです。<sup&gt; 1</sup&gt;。これらの複雑な細胞小器官もアポトーシス細胞死の重要な役割を果たして<sup&gt; 2</sup&gt;、細胞の生存<sup&gt; 3</sup&gt;、哺乳類の開発<sup&gt; 4</sup&gt;、ニューロンの発達と機能<sup&gt; 4</sup&gt;、細胞内シグナル伝達<sup&gt; 5</sup&gt;、および寿命調節<sup&gt; 6</sup&gt;。ミトコンドリアによって制御されるこれらの複雑な生物学的プロセスについての我々の理解は、その形態、それらのタンパク質と脂質組成、彼らのDNAの完全性、およびそれらの数多くの重要な機能を評価するための堅牢な方法に依存しています。出芽酵母<em&gt;サッカロマイセスセレビシエ</em&gt;、注釈付きのゲノムと明確に定義されたプロテオームとの遺伝的および生化学的に操作可能な単細胞真核生物は、ミトコンドリアの本質的な生物学的機能の根底にある分子細胞メカニズムを研究するための貴重なモデルです。このような種類の勉強のために、それは高純度のミトコンドリアを持つことが重要です。ここでは、酵母ミトコンドリアの精製のための迅速かつ効果的な方法の詳細な説明を提示する。このメソッドは、他の細胞小器官による汚染の本質的に自由であり、それらの精製後、構造と機能の整合性を維持する高純度のミトコンドリアの分離が可能になります。この方法で精製したミトコンドリアは、本質的なミトコンドリアのプロセスの無細胞再構成に適しており、ミトコンドリアの構造と機能、ミトコンドリアプロテオームとlipidome、およびミトコンドリアDNAの解析に使用することができます。

Watch this Scientific Journal Video about Purification of Mitochondria from Yeast Cells at JoVE.com

Purification of Mitochondria from Yeast Cells

我々は、酵母からのミトコンドリアの精製のための迅速かつ効果的な方法を説明します<em&gt;サッカロマイセスセレビシエ</em&gt;。このメソッドは、他の細胞小器官による汚染の本質的に自由であり、それらの精製後、構造と機能の整合性を維持する純粋なミトコンドリアの高収量を可能にします。

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 ...

purification-of-mitochondria-from-yeast-cells

Research

JoVE Journal

Biology

25.0K ビュー.  Concordia University. 我々は、酵母からのミトコンドリアの精製のための迅速かつ効果的な方法を説明しますサッカロマイセスセレビシエ。このメソッドは、他の細胞小器官による汚染の本質的に自由であり、それらの精製後、構造と機能の整合性を維持する純粋なミトコンドリアの高収量を可能にします。

ビデオ: Purification of Mitochondria from Yeast Cells

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

CD4 +ミルテニーMACS精製を使用してマウスのリンパ節からT細胞の分離

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

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 ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

高活性酵母のリボソームのクロマトグラフィー精製

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

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 ...

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes&#8217; association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions&#160;in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

ローカライズされた翻訳のメカニズムを研究するために酵母ミトコンドリアでのmRNA関連のアイソレーション

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

組織解離と差動濾過により移植のためにミトコンドリアの迅速な単離および精製

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

培養細胞およびマウス組織から単離とミトコンドリアの機能解析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

真核細胞から体外の環状DNAのゲノムワイドな精製

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Purification of Platelets from Mouse Blood

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), and plasma proteins. We describe here purification of platelets from mouse blood using three-fold more iohexol gradient medium relative to blood sample volume and centrifugation in a swinging bucket rotor at 400 x g for 20 min at 20 &#176;C. The recovery/yield of the purified platelets were 18.2-38.5%, and the purified platelets were in a resting state, which did not contain any significant number of RBC and WBC. The purified platelets treated with thrombin showed up to 93% activation, indicating their viability. We confirmed that the purified platelets are sufficiently pure using flow cytometric and microscopic evaluation. These platelets can be used for gene expression, activation, granule release, aggregation, and adhesion assays. This method can be used for purification of platelets from the blood of other species as well.

Here, mouse blood was collected in the presence of an anti-coagulant. The platelets were purified by iohexol gradient medium using low speed centrifugation. The platelets were activated with thrombin to investigate if they were viable. The quality of the purified platelets was analyzed by flow cytometry and microscopy.

マウスの血液からの血小板の浄化

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), ...