国立衛生研究所、NHLBI（RD、PY、PS）。 米国心臓協会（JL）。 スタンフォード大学、VPUEグラント（JL）。

事前調査1から適応した。 1。 SPIOのアネキシンVの結合<ol><li> 2：0.15 M過ヨウ素酸ナトリウム（過ヨウ素4）（重量比4:1の重量）と溶液中で暗所で20℃で1時間SPIO粒子（オーシャンナノテク社は、5 mg / mL）を°Cを酸化させる。 </li><li> 20℃で0.15 Mホウ酸ナトリウムた（Na 2 B 4 O 7 10H 2 O）で：ANXタンパク質を（重量比1:1、重量）を精製して12時間酸化SPIOをインキュベート</li><li>ヒュームフードの下水素化ホウ素ナトリウム（NaBH 4の ）で1時間さらに混合物を削減し、その後、0.15Mトリス-HClで急冷する。 </li><li> 14,000×gで（75μmの細孔マイクロコンフィルターミリポア、ビルリカ、MA）で遠心分離してANX-SPIOから無料でANXを分離します。 </li><li>さらに磁気分離装置（オーシャンナノテク、（株））を使用してバインドされていない不純物を除去することによって化合物を絞り込む。 </li><li>フィルター残留（ANX-SPIO）タンパク質レベルを定量化する-80 Bradfordタンパク質アッセイ、店舗によって2℃で1％血清アルブミンおよびプロテアーゼ阻害剤（HALTプロテアーゼインヒビター、サーモサイエンティフィック、1から50ミリグラム/ mgでANX）の0.1 Mトリス-HCl mg / mLの。 </li></ol> 2。動的光散乱<ol><li>別々に0.1の光学密度になるようにPBSでSPIOとANX-SPIOを希釈し、ゼータサイザーナノDLSマシン（マルバーン社、ウスターシャー州、イギリス）3動的光散乱により解析する。 </li><li> Z-平均粒径と多分散性指数（PDI）と、化合物の単峰性のピークを（理想的）を取得します。単独で酸化鉄ナノ粒子のDLS-測定サイズにこのサイズを比較します。 ANX-SPIO DLSの結果は、同種のANX-SPIO種3を反映して、0.13 1のPDIの78 nmの共役粒子流体力学的直径を示した。 </li></ol> 3。細胞培養、培養中のアポトーシスの誘導、培養細胞のMRI <ol><li>文化成体マウス骨髄間葉系のDMEMのTEM細胞（のMMSC）は、好ましくは、通路20から30まで4℃、10％FBSを添加し、1％ペニシリン-ストレプトマイシン（インビトロジェン社）。 </li><li> DOX（Sigma社、0.9％生理食塩水に1 mMのは、メディアに1μMの濃度）を12に37℃で時間を変化させるために処理することにより、培養細胞にアポトーシスを誘導する。 </li><li> ANX-SPIO（下記の3.4で説明）とMRIやTUNEL染色後、顕微鏡を用いた（APO-BrdUTM TUNELアッセイキット、Molecular Probes社/ Invitrogen社、ユージーン、OR）、またはANX-FITCで染色した後のいずれかを使用して、培養中のアポトーシスの量を定量（Roche社、スイス）。 </li><li> 37℃15分℃のため、ANX-SPIO（特に指定のない限りは10mgタンパク質/ mLの培地）で、または無料のSPIO（5 mg）の当量で細胞を標識SPIOナノ粒子の拡散を防ぐために、1％アガロース、1 mLのPBSで4回と、オーバーレイを洗います。料理のMRI（後述）を実行します。あるいは、細胞を洗浄し、DOXとANX-SPIO標識後トリプシン処理し、細胞ができるようにエッペンドルフチュー​​ブにペレットを形成しています。 </li></ol> 4 体外 MRI で <ol><li> in vitroで MRI分析のための標準的な膝の受信コイル1.5または3テスラGE SignaのEXCITEスキャナを使用しています。 </li><li>寒天ファントムに培養皿やチューブを配置し、勾配エコー甘やかされて育ったグラデーションのシーケンスを使用してイメージ：繰り返し時間（TR）550ミリ秒、エコー時間（TE）を10 ms、フリップ角15°、マトリクス256×256、フィールドのビュー3センチメートル×3 cmのスライス厚1.3ミリメートル、平均回数（NEX）2、間隔0ミリメートル5。固定領域（例えば、0.8ミリメートル2）地域·オブ·インタレスト（ROIを）を使用して、T2 *信号損失のためのプレート上の細胞層を介して軸方向に画像。寒天ファントムからの制御ROIの信号を使用して背景を調整します。ファントムを周囲の空気の信号からバックグラウンドノイズを測定します。 </li><li> [SIサンプルROI-SI制御ROI] / SIは信号強度で、[バックグラウンドノイズのSIのSD]と：MRIコントラスト対ノイズ比（CNRを計算するSDは標準偏差である）各培養皿を算出した。 </li></ol> 5。代表的な結果 in vitroでの T2 *信号損失の測定は、同種の寒天ファントムおよびシグナルボイドアーティファクトを作成する気泡の欠如を必要とします。このアーティファクトはSPIOの本物のT2 *信号損失と区別することは困難であり、解釈結果に非常に重要です。空気のアーティファクトと同様に本物の酸化鉄T2 *信号損失の例については、 図1を参照してください。 in vitroでの培養皿イメージングのために、厚さ約1 mmである十分な軸方向のMRIスライスは、適切に画像全体の料理をさせることができるように、各培養皿に寒天ゲルの適切な量を適用します。 図2は、予想されるMRI信号を示しています寒天内の個々の培養プレートから。プレートの端でいくつかの信号変化は、int型の領域のように分析、典型的にありますerestは、これらの不均一な領域を避ける必要があります。 <img alt="図1" src="/files/ftp_upload/3775/3775fig1.jpg" /> 図1：ANX-SPIOは、培養新生児ラット心筋細胞のアポトーシスを検出します。 図1Aは、制御ソリューション（左チューブ）ANX-SPIO（中間管）、およびDOX + ANX-SPIO（右チューブ）で処理してペレット化した新生児の心筋細胞の写真を示しています。 。アポトーシス細胞におけるANX-SPIO化合物の増加の保持を反映して、DOX + ANX-SPIO処理した細胞の褐色がかった色に注意してください。 図1B は 、in vitro MRI画像では、DOXの強烈なT2 *信号損失+ ANX-SPIO細胞を示しています。また、気泡は、4 番目と信号ANX-SPIOのvoid、およびCNRの分析に影響を与えることに似ています第 5 回エッペンドルフチューブ（小さ ​​な黒信号ボイド）の間に見られている。 <img alt="図2" src="/files/ftp_upload/3775/3775fig2.jpg" /> 図2：ANX-SPIO結合は、培養中のアポトーシス細胞に特異的である。 図2Aは、ANX-SPIOの低および高濃度（それぞれ、左と右）ANX-FLUOS染色またはANX-SPIO染色のいずれかに受け、培養皿上でアポトーシスラット新生児心筋細胞を、示しています。蛍光ANX（ANX-FLUOS）とANX-SPIOの高レベルと低レベルで処理したアポトーシス細胞の異なったT2 *信号損失（黒信号）に注意してください。右側に増加したT2 *信号の損失は、より高いASX-SPIOの濃度ではオフに出場された著しく減少ANX-FLUOS信号に関連付けられています。 図2Bは、ANX-SPIO対ANX-FLUOS信号と解離の競合的結合を示しています定数は、i K。 X軸は対数スケールでのアネキシン蛋白質濃度を示しています。 ムービー1。 <a href="http://www.jove.com/files/ftp_upload/3775/3775movie1.mov" target="_blank">補足表示するには、ここをクリック</a>映画。

<ol>
	<li>Dash, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+MRI+probe+to+detect+treatment+of+cardiac+apoptosis+in+vivo.">A molecular MRI probe to detect treatment of cardiac apoptosis in vivo.</a> Magn. Reson. Med. , (2011).</li><li>Khym, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reaction+of+methylamine+with+periodate-oxidized+adenosine+5'-phosphate.">The reaction of methylamine with periodate-oxidized adenosine 5'-phosphate.</a> Biochemistry. 2, 344-350 (1963).</li><li>Liu, Y. Y., Yu, Y., Zhang, G. B., Tang, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+characterization,+and+controlled+release+of+novel+nanoparticles+based+on+MMA/beta-CD+copolymers.">Preparation, characterization, and controlled release of novel nanoparticles based on MMA/beta-CD copolymers.</a> Macromol. Biosci. 7, 1250-1257 (2007).</li><li>Seo, 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=FeCo/graphitic-shell+nanocrystals+as+advanced+magnetic-resonance-imaging+and+near-infrared+agents.">FeCo/graphitic-shell nanocrystals as advanced magnetic-resonance-imaging and near-infrared agents.</a> Nat. Mater. 5, 971-976 (2006).</li><li>Hsiao, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+nanoparticle+labeling+of+mesenchymal+stem+cells+without+transfection+agent:+cellular+behavior+and+capability+of+detection+with+clinical+1.5+T+magnetic+resonance+at+the+single+cell+level.">Magnetic nanoparticle labeling of mesenchymal stem cells without transfection agent: cellular behavior and capability of detection with clinical 1.5 T magnetic resonance at the single cell level.</a> Magn. Reson. Med. 58, 717-724 (2007).</li><li>Zhao, M., Beauregard, D. A., Loizou, L., Davletov, B., Brindle, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+detection+of+apoptosis+using+magnetic+resonance+imaging+and+a+targeted+contrast+agent.">Non-invasive detection of apoptosis using magnetic resonance imaging and a targeted contrast agent.</a> Nat. Med. 7, 1241-1244 (2001).</li><li>van Tilborg, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internalization+of+annexin+A5-functionalized+iron+oxide+particles+by+apoptotic+Jurkat+cells.">Internalization of annexin A5-functionalized iron oxide particles by apoptotic Jurkat cells.</a> Contrast Media Mol. Imaging. 4, 24-32 (2009).</li><li>Sosnovik, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+cardiomyocyte+apoptosis+with+a+novel+magneto-optical+nanoparticle.">Magnetic resonance imaging of cardiomyocyte apoptosis with a novel magneto-optical nanoparticle.</a> Magn. Reson. Med. 54, 718-724 (2005).</li><li>Sosnovik, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+MRI+detects+low+levels+of+cardiomyocyte+apoptosis+in+a+transgenic+model+of+chronic+heart+failure.">Molecular MRI detects low levels of cardiomyocyte apoptosis in a transgenic model of chronic heart failure.</a> Circ. Cardiovasc. Imaging. 2, 468-475 (2009).</li><li>Dumont, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+death+induced+by+myocardial+ischemia+and+reperfusion:+measurement+with+recombinant+human+annexin-V+in+a+mouse+model.">Cardiomyocyte death induced by myocardial ischemia and reperfusion: measurement with recombinant human annexin-V in a mouse model.</a> Circulation. 102, 1564-1568 (2000).</li><li>Blankenberg, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+detection+and+imaging+of+phosphatidylserine+expression+during+programmed+cell+death.">In vivo detection and imaging of phosphatidylserine expression during programmed cell death.</a> Proc. Natl. Acad. Sci. U.S.A. 95, 6349-6354 (1998).</li><li>Schellenberger, E. A., Sosnovik, D., Weissleder, R., Josephson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magneto/optical+annexin+V,+a+multimodal+protein.">Magneto/optical annexin V, a multimodal protein.</a> Bioconjug. Chem. 15, 1062-1067 (2004).</li></ol>

利害の衝突が宣言されません。

アネキシンVにSPIOをリンクするための説明した接合方法は、アネキシンとSPIOナノ粒子のカルボキシル部分の側鎖アミン基を利用しています。特定の酸化還元工程を経て、これらの化合物の共有結合を達成することができ、得られた官能性ナノ粒子を単離することができる。このメソッドは、興味のある他のタンパク質とナノ粒子に一般化することができる。 最も重要な?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ＃ </td></tr><tr><td>超常磁性酸化鉄</td><td>オーシャンナノテク株式会社</td><td> ICK-40から005 </td></tr><tr><td>ドキソルビシン</td><td>シグマ</td><td> D1515-10mgの</td></tr><tr><td>セパレータをSuperMag </td><td>オーシャンナノテク株式会社</td><td> N / A </td></tr><tr><td>ゼータサイザーナノDLSマシン</td><td>マルバーン社</td><td> N / A </td></tr></tbody></table>

synthesis of an in vivo mri-detectable apoptosis probe

細胞のアポトーシスは、多くの疾患の顕著な特徴であり、疾患の臨床症状が明らかになる前にこのプログラム細胞死は、通常発生します。その初期、可逆段階でアポトーシスを検出する手段が予防または治療措置が永久的な損傷から心臓を保護するために取ることができる、その間に前臨床試験 &#39;ウィンドウを&#39;余裕でしょう。我々はここにMRIで検出可能な造影剤として機能する超常磁性酸化鉄（SPIO）粒子に熱心にアポトーシスの最も初期の、可逆的な段階で細胞に結合するヒトアネキシンV（ANX）を、共役するためのシンプルかつ堅牢な方法を提示する。接合方法は、SPIOの多糖類のシェルにカルボキシル基を酸化しSPIOナノ粒子の酸化で始まります。 ANXタンパク質を精製した後の削減バッファにSPIOとANXの共有結合性相互作用を容易にするために、ホウ酸ナトリウム溶液の設定で追加されます。ナトリウムborohydrid最終的な還元工程eは削減を完了するために実行され、その後反応がクエンチされる。非抱合ANXは、遠心濾過により混合物から除去されます。 ANX-SPIO製品のサイズと純度は、動的光散乱（DLS）によって検証されます。このメソッドは、架橋された酸化鉄粒子の共役メソッドまたはビオチン標識ナノ粒子とは対照的に、への追加を必要とする、または多糖類SPIOシェルの変更はありません。結果として、このメソッドは、興味のある他のタンパク質の結合に延長することができる、シンプルで堅牢なアプローチを表しています。

Watch this Scientific Journal Video about Synthesis of an In vivo MRI-detectable Apoptosis Probe at JoVE.com

Synthesis of an In vivo MRI-detectable Apoptosis Probe

アポトーシスの早期発見は病気の様々なリスクの高い細胞集団を識別することができる。ここでは、MRIで検出可能な酸化鉄ナノ粒子（SPIO）に初期アポトーシス検出タンパク質（アネキシンV）にリンクする方法を示しています。このメソッドは、MRIで検出可能な分子イメージングプローブを生成するために関心のある他のタンパク質に延長することができる。

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 ...

synthesis-of-an-in-vivo-mri-detectable-apoptosis-probe

Research

JoVE Journal

Biology

Synthesis of an In vivo MRI-detectable Apoptosis Probe

9.1K ビュー.  Stanford University Medical Center. アポトーシスの早期発見は病気の様々なリスクの高い細胞集団を識別することができる。ここでは、MRIで検出可能な酸化鉄ナノ粒子（SPIO）に初期アポトーシス検出タンパク質（アネキシンV）にリンクする方法を示しています。このメソッドは、MRIで検出可能な分子イメージングプローブを生成するために関心のある他のタンパク質に延長することができ...

ビデオ: Synthesis of an In vivo MRI-detectable Apoptosis Probe

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

全プラスミドの自家製のサイトが特異的突然変異誘発

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

生物学

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

遺伝的にコードされたカルシウムのセンサを用いたアポトーシス時のミトコンドリア内カルシウム濃度の動的変更の監視

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios &lt;0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (&lt; 5% events with cytoplasmic PI staining).

An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.

細胞死の正確な評価のために修飾されたアネキシンV /ヨウ化プロピジウムのアポトーシスアッセイ

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

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 ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

モザイク大人のライブイメージングのためのトマト/ GFP-FLP / FRT方法<em&gt;ショウジョウバエ</em&gt;視細胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

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 ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

アン in vivoで</emからのタンパク質複合体を分離するために>架橋アプローチショウジョウバエ胚

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

アン<em&gt;生体外</em甲状腺の開発を研究する&gt;文化システム

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

ポリソームプロファイリングによる哺乳動物細胞における選択的mRNAの翻訳の評価

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...