この作品は、MDにNIHの助成金NS042197によってサポートされていました。 AJKは、助成金T32GM008719とF30NS068006によってサポートされていました。

1。マイクロインジェクションの針の生産<ol><li>プレハブマイクロインジェクションの針は、市販されている（ 例えば 。エッペンドルフからFemtotips）と一microinjectionsの多数実行されていない場合に便利です。しかし、マイクロインジェクションのために長期的な能力を確立したい人のために、代替手段は薄い壁ホウケイ酸ガラス毛細管と商業ニードルプラーを使用して実験室でマイクロインジェクションの針を生成することです。これはまた、針の形状を変化させることができますさまざまな種類の細胞に有用であることができる。 </li><li>ナリシゲPC - 10マイクロインジェクションニードルプラーで、すべての4つの重みを添付し、58.0（第2のヒーター）で相対的な熱の設定でワンステップ引っ張るプログラム（ステップ1の設定）を使用します。結果の2つの針が同じような長さになるように各キャピラリーの中心に発熱体を配置してください。 </li><li>いくつかの毛細血管を（関心の各タンパク質の約2毛細血管）を引き出します。 </li><li>針の先端を破損しないようにしている間に容器に針を保管してください。このような発泡体またはBlu -タックなどの材料は、マイクロインジェクションの針を保持するためにコンテナで使用することができます。 </li></ol> 2。注射用タンパク質混合物の調製<ol><li> 1 M塩化カリウム及びpH7.4の0.1 Mリン酸カリウム（KPI）バッファ（K 2 HPO 4、KH 2 PO 4の等モル混合物）を含む10倍マイクロインジェクションのバッファを準備します。このバッファは、室温での長期保存することができます。 </li><li>注射を可視化するために、ローダミン-デキストランのような蛍光色素を追加する必要があります。水中でthe10xマイクロインジェクションバッファーを希釈し、20〜40 mg / mLのローダミンデキストランを含む5倍のマイクロインジェクション緩衝液を作るためにローダミン-デキストラン粉末を溶解する。 </li><li> 4℃暗所でこのソリューション℃にて保存してください。それは、最大100個々のタンパク質溶液製剤の十分なはずです。フルオレセインイソチオシアネート - デキストランのような他の染料は、置き換えることができます。 </li><li> 20 mg / mLの濃度に水で精製シトクロム c を溶解することによってシトクロム c の株式を準備します。 -80℃ストアシトクロム c ° Cの長期保管と小さい（〜10μL）アリコートにシトクロム c を格納することにより、凍結融解を避けるため。 </li><li> 3μLの水と1 ×緩衝液（100mM KCl、10mMのKPに10 mg / mLのシトクロム cの最終濃度は5μL シトクロム cとローダミン-デキストランを含む2μL5倍のマイクロインジェクションのバッファを組み合わせることにより、注射用10μLのタンパク質混合物を準備する私 、4-8 mg / mLのローダミンデキストラン）。 </li></ol> 3。 シトクロム cの細胞質マイクロインジェクション<ol><li>直前にマイクロインジェクションに、℃のマイクロインジェクションの針を詰まらせる可能性のある粒子状物質を分離するために4℃で10分間16,000 gでシトクロム cおよびマイクロインジェクションバッファーの混合物を遠心分離。 </li><li>遠心操作中に、空気の圧力が構築できるようにするインジェクターをオンにします。 </li><li>顕微鏡ステージの中央に細胞の皿を置き、セルにフォーカスを設定します。細胞をインキュベーター外で保持される時間の量を最小限に抑えるために、細胞は通常、30分以内にインキュベーターに戻されます。 </li><li>キャピラリーの平滑末端へのタンパク質の混合物の上面からピペット0.5から1μL。チューブの底に遠心分離されているすべての粒子をピペッティングしないように注意してください。分以内に、タンパク質混合物は毛細管現象を介して針の先端に配布します。 </li><li>マイクロマニピュレータと位置の先端が約45 °の角度で、顕微鏡の透過光を通過するように針のキャピラリーホルダーにしっかりと針を取り付けます。 </li><li>それは視野の中​​心に直接置かれているように、針の先端の位置を調整します。センター針をするために、顕微鏡の接眼レンズをのぞきながら、針を移動するためにマイクロマニピュレーターを使用してください。針の影が見えるはずです。その影が唯一の針先がセルの上に中央揃えされていることを示す、視野の半分に見られるようにニードルを調整します。 </li><li>連続フローモードにインジェクターを設定し、20に作動圧力を​​設定する - 100 hPaのを。それぞれの針は、別の作動圧力を​​必要とするかもしれません、と使用圧力は、おそらく一定の流れを維持するためにマイクロインジェクションの手順の間に調整が必要になります。 </li><li>ちょうど細胞上に位置する粗ノブを使って針を下ろします。これを行うには、ちょうど細胞上の位置に顕微鏡の焦点面を上げる。針の先端にフォーカスがあるまで、粗いノブを使用してセルに向かって針を下ろします。 </li><li>再中央針の視野内の先端と顕微鏡対物を変更することにより、倍率を増加させる。 </li><li>徐々に細かいマニピュレータノブuntiを使用して針を下げるlは針がわずかに細胞の焦点面より上です。 </li><li>ローダミンの赤色の蛍光を見ることによって、タンパク質の混合物の流れを確認してください。タンパク質混合物は薄い、定数ストリームとして針を終了する必要があります。針が危険にさらされている場合またはフローがあまりにも強い場合に針を交換して再ロードする必要があります。時には、ガラスキャピラリーの先端はクローズされ、フローが針から見られない。この問題が発生した場合は、針を交換するか、または慎重にゆっくりと破裂針の先端に培養皿の底にそれを下げる。 </li><li>それは約45 °の角度でセル方向を向いているように、針の先端を合わせます。セルに向かって移動しながらの滑らかな動きで、針を下ろします。第二滑らかな動きで、すぐに細胞からそれを削除するには、針の方向を反​​転させます。 </li><li>が正常に注入された細胞は、しばしばわずかに膨潤し、細胞内の赤色蛍光ローダミンを視覚化することによって確認することができます。時折、セルが誤って表示される核に注入されます。 </li><li>約50から100の細胞が注入されるまで、顕微鏡のステージを調整することによって、細胞を注入し続ける。顕微鏡ステージを移動するとき、それは細胞の上部をクリアするようにマイクロインジェクションの針を上げてください。 </li></ol> 4。代表的な結果： シトクロム cの細胞質マイクロインジェクションは、アポトーシス中にミトコンドリアから放出を模倣しています。このように、予想通り、線維芽細胞は急速にウシシトクロム c（図1A）の細胞質マイクロインジェクションによりアポトーシスを起こす。酵母シトクロム c はカスパーゼ12を活性化することはできないので単独で注射の手順は、細胞死の責任ではないことを保証するために、酵母シトクロム cの注入は、重要なコントロールとして機能します。 興味深いことに、有糸分裂後交感神経ニューロンは、細胞質シトクロム c（図1B）8,13に対して非常に耐性があります。私たちの研究室では、内因性カスパーゼ阻害剤のXIAPは、ニューロン14のカスパーゼ活性化の鍵阻害剤であることを確認しています。したがって、 シトクロム c の注射後に死ぬためにニューロンは、XIAPは、まず不活性化する必要があります。例えば、XIAPへのシトクロム cのマイクロインジェクション- / -交感神経ニューロンは、これらの細胞におけるカスパーゼ活性化とアポトーシス（図2）を可能にする十分です。 <img src="/files/ftp_upload/2773/2773fig1.jpg" alt="図1"/> 図1。 シトクロム cの細胞質マイクロインジェクションは、急速に線維芽細胞の死ではなく、神経細胞を誘導する。 A）野生型MEFには、または（B）生後5日目の野生型交感神経ニューロンは、注入された細胞をマークするためにローダミン-デキストランと一緒にウシシトクロム c（に10 mg / mL）でマイクロインジェクションした。画像はセルの同じフィールドの直後に注射（0時間）を表示、または指定された時間に。矢印は、注入された細胞を示す。スケールバー、20μmである。 <img src="/files/ftp_upload/2773/2773fig2.jpg" alt="図2"/> 図2。 XIAP欠損ニューロンは、細胞質シトクロム c のマイクロインジェクションの影響を受けやすくなっています 。 XIAPノックアウトマウスから生後5日目交感神経のニューロンは、注入された細胞をマークするためにローダミン-デキストランと一緒にウシシトクロム c（に10 mg / mL）でマイクロインジェクションした。画像は、同じ細胞をすぐに次の注入のフィールド（0時間）、またはシトクロム c のマイクロインジェクション後の5時間（5時間）を示す。スケールバー、20μmである。

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動物のすべての実験手順は、ノースカロライナ大学の動物実験使用の委員会によって承認された。

直接細胞の細胞質へのシトクロム cのマイクロインジェクションは、アポトーシスの後のミトコンドリア調節の研究を可能にするユニークで強力なツールです。重要なのは、この手法は、細胞やミトコンドリアの損傷を引き起こす薬剤を使用せずに、ミトコンドリアのアポトーシスの下流の直接活性化することができます。 このプロトコルは、アポトーシスの研究...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td> DM IRE2倒立顕微鏡</td><td>ライカ</td><td></td><td></td></tr><tr><td> PC - 10マイクロインジェクションニードルプラー</td><td>ナリシゲ</td><td></td><td></td></tr><tr><td> MWO - 202マニピュレーター</td><td>ナリシゲ</td><td></td><td></td></tr><tr><td> FemtoJetマイクロインジェクター</td><td>エッペンドルフ</td><td></td><td></td></tr><tr><td>ミクロフィラメントと薄肉Boroscilicateガラスキャピラリー</td><td> AMシステム</td><td> 615000 </td><td> 4インチの長さは、1.00ミリメートル、外径、0.75ミリメートル、内径</td></tr><tr><td>ローダミンBイソチオシアネート - デキストラン</td><td>シグマアルドリッチ</td><td> R9379 </td><td>平均分子量〜70,000ダ</td></tr><tr><td>ウシシトクロム c蛋白質</td><td>シグマアルドリッチ</td><td> C3131 </td><td></td></tr></tbody></table>

activation of apoptosis by cytoplasmic microinjection of cytochrome c

アポトーシス、またはプログラムされた細胞死は、細胞が1を死滅することによって保存し、高度に制御された経路である。細胞は細胞傷害性ストレスの広い範囲に到達したときにアポトーシスを引き起こすことができます。これらの侮辱は、最終的にミトコンドリアの膜間腔から細胞質2〜 シトクロム cの放出を引き起こすシグナル伝達カスケードを開始する。ミトコンドリアからのシトクロム cの放出は、カスパーゼ、最終的に3〜4細胞死を実行する主要な細胞内プロテアーゼの急速な活性化をトリガーするキーイベントです。 アポトーシスの経路はミトコンドリア5からのシトクロム c の放出の上流と下流のポイントで規制されている。カスパーゼ活性化の後のミトコンドリア調節を研究するためには、多くの研究者は細胞6月9日にholocytochrome C（ヘムに接続された）タンパク質の細胞質マイクロインジェクションを指示することになっている。 シトクロム c は、通常、ヘムグループの添付ファイルはそれが10月11日アポトーシス活性化できるようにするために必要であるミトコンドリアに局在している。したがって、直接カスパーゼを活性化するために、それはcDNA構築物からシトクロム cの発現がミトコンドリアターゲティングとヘムの添付ファイルにつながる一方、それは細胞質カスパーゼから隔離されるため、holocytochrome Cタンパク質の代わりに、そのcDNAを注入する必要があります。このように、精製したヘムに接続されたシトクロム c 蛋白質の直接細胞質マイクロインジェクションは、細胞とミトコンドリアの損傷を引き起こす有毒な侮辱を使用せずに、ミトコンドリアのシトクロム c の放出とアポトーシスを模倣するために有用なツールです。 この記事では、我々はマウス胚性繊維芽細胞（MEF）と例のような主要な交感神経ニューロンを用いて、細胞内へのシトクロム c 蛋白質のマイクロインジェクションのための方法を説明します。このプロトコルは、アポトーシスの調査のためのシトクロム cの注入に焦点を当てているが、ここに示されている技術も容易に関心のある他のタンパク質のマイクロインジェクションのために適応させることができる。

Watch this Scientific Journal Video about Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c at JoVE.com

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

このプロトコルでは、我々は、シトクロムの直接細胞質マイクロインジェクションを説明します<em&gt; C</em線維芽細胞と一次交感神経ニューロンへ&gt;タンパク質。このテクニックは、シトクロムの導入が可能<em&gt; C</em細胞とミミックシトクロムの放出の細胞質へ&gt;タンパク質<em&gt; C</em&gt;ミトコンドリアから、これは、アポトーシス中に発生します。

シトクロムの細胞質マイクロインジェクションによるアポトーシスの活性化 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

activation-of-apoptosis-by-cytoplasmic-microinjection-of-cytochrome-c

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Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

17.3K ビュー.  University of North Carolina. このプロトコルでは、我々は、シトクロムの直接細胞質マイクロインジェクションを説明します Cタンパク質。このテクニックは、シトクロムの導入が可能 Cタンパク質 Cミトコンドリアから、これは、アポトーシス中に発生します。

ビデオ: Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques.   In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.  Central to these studies is CD1d - the antigen presenting molecule that presents glycolipids to NKT cells.  The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells.  Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy.  Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmunity, clearance of infection, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.

インタビュー：CD1dによる糖脂質抗原提示とNKT細胞の活性化の治療の可能性

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious ...

生物学

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

マイクロインジェクションを使用して安定したトランスジェニック線虫の世代

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

トランスジェニックの世代 C.エレガンスバイオリスティック変換による

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

マイクロインジェクションによる哺乳動物細胞におけるmRNA核外輸送の速度論の解析

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

偏上皮細胞へのプラスミドのマイクロインジェクションによる低分子量GTPaseの機能を解析する

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

シトクロムを使用してミトコンドリア呼吸機能の可視化 Cオキシダーゼ/コハク酸脱水素酵素（COX / SDH）二重標識組織化学

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are evident. A means to detect apoptosis in its earliest, reversible stages would afford a pre-clinical 'window' during which preventive or therapeutic measures could be taken to protect the heart from permanent damage. We present herein a simple and robust method to conjugate human Annexin V (ANX), which avidly binds to cells in the earliest, reversible stages of apoptosis, to superparamagnetic iron oxide (SPIO) nanoparticles, which serve as an MRI-detectable contrast agent. The conjugation method begins with an oxidation of the SPIO nanoparticles, which oxidizes carboxyl groups on the polysaccharide shell of SPIO. Purified ANX protein is then added in the setting of a sodium borate solution to facilitate covalent interaction of ANX with SPIO in a reducing buffer. A final reduction step with sodium borohydride is performed to complete the reduction, and then the reaction is quenched. Unconjugated ANX is removed from the mix by microcentrifuge filtration. The size and purity of the ANX-SPIO product is verified by dynamic light scattering (DLS). This method does not require addition to, or modification of, the polysaccharide SPIO shell, as opposed to cross-linked iron oxide particle conjugation methods or biotin-labeled nanoparticles. As a result, this method represents a simple, robust approach that may be extended to conjugation of other proteins of interest.

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes.

の合成<emインビボ&gt;</em&gt; MRIで検出可能なアポトーシスプローブ

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

遺伝子調節の1分子蛍光イメージング<emインビボ&gt;</em&gt;開裂により同時翻訳アクティベーションの使用（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

活性化カスパーゼ3を検出するために、ホールマウント免疫蛍光法によりゼブラフィッシュ胚におけるアポトーシスの分析

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA (dsDNA) which has the requisite characteristics to stimulate the cytoplasmic DNA sensing pathways can be generated cheaply and with ease. By the concatemerization of short, synthetic oligonucleotides (which lack CpG motifs), dsDNA can be generated to be of sufficient length to activate the cytosolic DNA sensing pathway. This protocol involves blunt end ligation of the oligonucleotides in the presence of polyethylene glycol (PEG), which provides an environment for efficient ligation to occur. The dsDNA concatemers can be used, following purification by phenol/chloroform extraction, to simulate the innate immune response in vitro by standard transfection protocols. This DNA can also be used to stimulate innate immunity in vivo by intradermal injection into the ear pinna of a mouse, for example. By standardizing the concatemerization process and the subsequent stimulation protocols, a reliable and reproducible activation of the innate immune system can be produced.

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

The aim of the protocol is to use optimal methods for stimulating the cytoplasmic DNA sensing pathways in cells and in vivo. This is achieved by improving the generation of long, double-stranded DNA during blunt-end ligation. Cells or mice are then transfected using a lipid transfection reagent.

細胞質DNAセンシング経路の刺激<em&gt;体外</em&gt;と<em&gt;インビボ</em

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...