失明を防ぐための研究、サンガー研究所、ウェルカムトラストゲノムキャンパスでとラミロ·ラミレス·ソリス、ジャッキーホワイト、ジャンヌEstabel; Bartly J·モンディーノMDは、ジュール·スタイン眼研究所、カリフォルニア大学ロサンゼルス校（UCLA）のディレクター。この研究は、眼科とビジュアルリサーチにおける動物の使用に関するARVOステートメントに準拠しています。

1。鈍的切開：未固定標本でマウス眼球摘出<ol><li>後部地球儀（眼球）表面への曝露とのアクセスを改善するためにまぶたを引き離す。 </li><li>軌道上の（下）地球儀（眼窩）の背後にある湾曲したドレッシング鉗子を置きます。マハジャンSharptipドレッシング鉗子は、（材料および試薬の表を参照）、この手順を容易にするために、先の尖った先端を持つカスタムインストゥルメントです。 </li><li>鉗子を閉じて、地球儀を圧迫しないように注意しながら、地球の背後にある軌道結合組織や視神経を把握する。 </li><li>ゆっくり上向きに鉗子を引くと軌道から眼球を摘み取る。白い糸状の組織が視神経です。 </li><li> PBSに目を置きます。 30ゲージの針を使用して、単一の穿刺は、眼球に針1〜2ミリメートルを挿入することにより、角膜輪部のすぐ後方に巻きつけてください。これは、眼組織に浸透することができませんそうでなければ、グルタルアルデヒドなどの固定剤は、、、エン直接することができますTER目。固定剤、例えばパラホルムアルデヒドやアルコールなど、関心の眼組織内に拡散することが可能である場合は、この刺し傷の作成は必要でないかもしれません。 </li><li>直ちに浸固定用固定液に全体の目を置きます。 </li></ol> 2。シャープ解剖：灌流固定後に、マウスの眼球摘出<ol><li>離れて地球からまぶたを保持するために湾曲したコリブリ鉗子を使用しています。 </li><li>地球に平行オリエント湾曲したウェストコット郭清をはさみ、軌道の背面方向を目指しながら。 </li><li>劣った、内側、優れており、側面から地球を囲む固定結合組織を切り取ります。 </li><li>地球の後ろに湾曲した鉗子を置き、世界中で押さずに結合組織をつかみ、目を摘出するために前方に引き出します。軌道の結合組織は、灌流固定から硬いですので、軌道組織のさらなる切断は完全にglobを解放する必要があるかもしれません電子。 </li></ol> 3。固定およびパッケージング固定組織の送料は、適切なラベルや包装も含めて制度や郵便サービス要件に従わなければなりません。次のプロトコルは、周囲温度で非感染性の生物学的サンプルを出荷した例です。これは、3包装の層が、ないクラスA / Bの生物学的物質または液体窒素ラベ​​ルを必要とします。 <ol><li>固定が短い場合、少なくとも3 mLの固定、またはポスト固定緩衝液で5 mLのガラス製シンチレーションバイアルに目を置きます。瓶の蓋の上に追跡番号を書いて、パラフィルムでそれを封印。 </li><li>生体試料中に置き、シンチレーションバイアルは、輸送用コンテナを承認した。サンプルが置かれている密封容器、吸収中間層、および保護外層：当研究所では、例えば、認定された容器は、3つの層を必要とします。 </li><li>バブルラップのパッケージにしてからアプリに容器を置きます適切なマニュ​​アルと一緒に実験室の輸送用コンテナをroved。 </li></ol> 4。代表的な結果目の組織学的検査をヘマトキシリン​​およびエオシン染色、酵素発現の検出、透過型電子顕微鏡、免疫組織化学を含む様​​々な方法により行うことができる。 4％パラホルムアルデヒド中で浸漬固定後、組織をミクロトームでパラフィン切片に埋め込まれていました。ハイスループット表現型では、瞳孔視神経節を分析し、その目の中のサンプルのすべての種類の組織（図1）。組織の切片もlacZ発現（図2）、細胞小器官の透過型電子顕微鏡（図3）、および抗体（図4）を持つ特定の分子の発現を調べた。 私たちの表現型スクリーニング方法では、（内側眼角）鼻と時間的（lateraに関して目の向きを維持することが重要ではありませんでしたリットル眼角）の側面。眼の向きが必要な場合は、少なくとも2つのオプションがあります。摘出した後、我々はハンドヘルド焼灼装置と3時の位置に一時的な角膜に光焼灼を適用している。病変は組織学的に明らかであり、組織学的にインクのような処理中に迷子になることはありません。欠点は、焼灼損害賠償審査のための重要な組織かもしれない角膜です。他のオプションは、外眼筋の挿入を識別することができる熟練した解剖学者を必要とします。どちら向きの方法で3劣ると上斜筋の挿入の識別が劣ると優れた、地球の側面にマークを付けます。実体顕微鏡下では、右または左眼等の標本を追跡することが重要です。一緒に、これは切断前に正確な地球のオリエンテーションを許容するでしょう。 <img src="/files/ftp_upload/3184/3184fig1.jpg" alt="図1" /> 図1。マウス眼組織学的画像はafte<html> Rリモート買収。マウスの目が4％パラホルムアルデヒドで灌流固定後に鋭い切開により摘出した。ヘマトキシリンとエオシンで染色Aの瞳孔視神経節では、すべての組織の下部構造の保全を示しています。視神経は、ON。 、IPL、内網状層、INLは、内顆粒層、OPL、外網状層、ONL、アウターRGC、網膜神経節細胞：正常な網膜の（緑のスケールバー= 500ミクロン）B.高倍率のビューには、層状構造を示しています核層（光受容細胞）（矢印）は、OS、感光体外側セグメント;、RPE、網膜色素上皮、C、脈絡膜、感光体の内側セグメント（矢頭）です。 （緑のスケールバー= 50ミクロン） では、通常の網膜に比べて、この試料は光受容体の変性にシンナーが原因です。 ONL、IS、およびOS（矢印）は完全に欠けている。 （緑のスケールバー= 50ミクロン） の &quot;alt =&quot;図2 &quot;/&gt; トランスジェニックマウスにおける図2 lacZ発現。目は（5mMのK3Fe（CN）6、5mMのK4Fe（CN）6、2 mMのMgCl2、0.02％NP40鈍的切開により摘出し、1 mg / mlのX-gal溶液に浸漬した、0.1％デオキシコール酸ナトリウム）を37℃で一晩目は30分、ミクロトームで切片は2％formaldehyde/0.2％グルタルアルデヒドで固定した。 X-galを積（青）ラベル：A.繊毛上皮、角膜上皮B.とC.網膜の網膜神経節細胞（RGC）層。 INLは、内顆粒層、ONL、外核層。 <img src="/files/ftp_upload/3184/3184fig3.jpg" alt="図3" /> 図3。網膜色素上皮（RPE）の透過型電子顕微鏡。刺し傷の鈍的切開および作成した後、目が浸いたが0.1Mリン酸ナトリウム緩衝液中の2.5％ホルムアルデヒド/ 2.5％グルタルアルデヒドで固定した。目はseconのあったdarily徐々に脱水し、1％オスミウムtextroxideで固定し、スパーの樹脂に包埋した。組織は90 nmであった切片とFormvarで覆われた銅スロットグリッド上に配置し、透過型電子顕微鏡を用いて撮像。顕微鏡写真は、感光体外側の神経感覚網膜のセグメントは、網膜色素上皮内メラノソーム（M）とブルッフ膜を示しています。 <img src="/files/ftp_upload/3184/3184fig4.jpg" alt="図4" /> 図4。マウスの硝子体中のスーパーオキシドジスムターゼ-3の免疫組織化学的標識（緑）。目は鈍的切開により摘出し、4％パラホルムアルデヒド中で浸漬固定を施行した。彼らは、ミクロトームを用いてパラフィン切片中に包埋した。組織切片を1:50に希釈したウサギポリクローナルSOD3抗血清とともにインキュベートした。 SOD3式はヤギ抗ウサギIgG（H + L）のAlexa Fluor 488標識二次抗体を用いて検出した。 SOD3の標識は、GREに示されている専用。

<ol>
	<li>Song, B. J., Tsang, S. H., Lin, C. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+models+of+retinal+degeneration+and+targets+for+gene+therapy.">Genetic models of retinal degeneration and targets for gene therapy.</a> Gene Ther. Mol. Biol. 11, 229-262 (2007).</li><li>Mahajan, V. B., Mondino, B. J., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+Mouse+Eye+Phenomics+System.">A high-throughput Mouse Eye Phenomics System.</a> Cold Spring Harbor Laboratories, Mouse Genetics Meeting. , (2010).</li><li>Smith, R. S., John, S. W. M., Nishina, P. M., Sundberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In Research Methods For Mutant Mice. , (2002).</li><li>Chang, B., Hawes, N. L., Hurd, R. E., Davisson, M. T., Nusinowitz, S., Heckenlively, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+mutants+in+the+mouse.">Retinal degeneration mutants in the mouse.</a> Vision Res. 42, 517-525 (2002).</li><li>Anderson, M. G., Smith, R. S., Hawes, N. L., Zabaleta, A., Chang, B., Wiggs, J. L., John, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+genes+encoding+melanosomal+proteins+cause+pigmentary+glaucoma+in+DBA/2J+mice.">Mutations in genes encoding melanosomal proteins cause pigmentary glaucoma in DBA/2J mice.</a> Nat. Genet. 30, 81-85 (2002).</li><li>Hawes, N. L., Smith, R. S., Chang, B., Davisson, M., Heckenlively, J. R., John, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+fundus+photography+and+angiography:+a+catalogue+of+normal+and+mutant+phenotypes.">Mouse fundus photography and angiography: a catalogue of normal and mutant phenotypes.</a> Mol. Vis. 5, 22-22 (1999).</li><li>Won, J., Shi, L. Y., Hicks, W., Wang, J., Hurd, R., Naggert, J. K., Chang, B., Nishina, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+resources+for+vision+research.">Mouse model resources for vision research.</a> J. Ophthalmol. 2011, 391384-391384 (2011).</li><li>Pinto, L. H., Vitaterna, M. H., Siepka, S. M., Shimomura, K., Lumayag, S., Baker, M., Fenner, D., Mullins, R. F., Sheffield, V. C., Stone, E. M., Heffron, E., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Results+from+screening+over+9000+mutation-bearing+mice+for+defects+in+the+electroretinogram+and+appearance+of+the+fundus.">Results from screening over 9000 mutation-bearing mice for defects in the electroretinogram and appearance of the fundus.</a> Vision. Res. 44, 3335-3345 (2004).</li><li>Heckenlively, J. R., Winston, J. V., Roderick, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+mouse+retinal+degenerations.+I.+Correlation+of+indirect+ophthalmoscopy,+electroretinograms,+and+histology.">Screening for mouse retinal degenerations. I. Correlation of indirect ophthalmoscopy, electroretinograms, and histology.</a> Doc. Ophthalmol. 71, 229-239 (1989).</li><li>Tsang, S. H., Gouras, P., Yamashita, C. K., Kjeldbye, H., Fisher, J., Farber, D. B., Goff, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+in+mice+lacking+the+gamma+subunit+of+the+rod+cGMP+phosphodiesterase.">Retinal degeneration in mice lacking the gamma subunit of the rod cGMP phosphodiesterase.</a> Science. 272, 1026-1029 (1996).</li><li>Tsang, S. H., Woodruff, M. L., Jun, L., Mahajan, V., Yamashita, C. K., Pedersen, R., Lin, C. S., Goff, S. P., Rosenberg, T., Larsen, M., Farber, D. B., Nusinowitz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+carrying+the+H258N+mutation+in+the+gene+encoding+the+beta-subunit+of+phosphodiesterase-6+(PDE6B)+provide+a+model+for+human+congenital+stationary+night+blindness.">Transgenic mice carrying the H258N mutation in the gene encoding the beta-subunit of phosphodiesterase-6 (PDE6B) provide a model for human congenital stationary night blindness.</a> Hum. Mutat. 28, 243-254 (2007).</li></ol>

特別な利害関係は宣言されません。

ほとんどのトランスジェニックマウスは、目を検査しません研究室に存在します。私たちのビデオ技術は目で少し経験を持つ研究室から組織の取り込みを最適化するために、遠隔手術技能移転のためのシンプルな、標準化された方法を示しています。この映像技術は、意味のある比較の表現型および分子生物学的研究を防止する非標準化組織採取と固定方法に起因専門サイトの限られた数の?...

<table border="1"><tbody><tr><th>名前</th><th>会社</th><th>カタログ番号</th><th>注釈</th></tr><tr><td>湾曲したドレッシング鉗子</td><td>シュトルツ眼科</td><td> E1408 </td><td></td></tr><tr><td>マハジャンSharptipドレッシング鉗子</td><td>シュトルツ眼科</td><td> E1406（REF SP7-64520） </td><td></td></tr><tr><td>湾曲したウェストコットはさみ</td><td>シュトルツ眼科</td><td> E3321 WH </td><td></td></tr><tr><td> 15°、BDビーバー顕微ブレイド</td><td>ベクトン·ディッキンソン</td><td> 374881 </td><td></td></tr><tr><td> 0.22ファインCastroviejo縫合鉗子</td><td>シュトルツ眼科</td><td> E1805 </td><td></td></tr><tr><td> 0.12コリブリ鉗子</td><td>シュトルツ眼科</td><td> 132分の2 </td><td></td></tr><tr><td> 30ゲージの針</td><td>ベクトン·ディッキンソン</td><td> 305128 </td><td></td></tr><tr><td>バイオハザードメーラー</td><td>フィッシャー</td><td> 03-523-4 </td><td></td></tr><tr><td>パラフィルム</td><td>フィッシャー</td><td> 13-374-10 </td><td></td></tr><tr><td>ガラスシンチレーションバイアル</td><td>ホイートン</td><td> 4500413033 </td><td></td></tr><tr><td> PBS（pH7.4）で</td><td>インビトロジェン</td><td> 70011-044 </td><td></td></tr><tr><td> 16％パラホルムアルデヒド</td><td>電子顕微鏡学</td><td> 15700 </td><td></td></tr><tr><td> 0.1Mリン酸ナトリウム緩衝液中の2.5％ホルムアルデヒド/ 2.5％グルタルアルデヒド</td><td>電子顕微鏡学</td><td> 15700＆16300 </td><td>実験室で混合</td></tr><tr><td> 50％のグルタルアルデヒド</td><td>電子顕微鏡学</td><td> 16300 </td><td></td></tr><tr><td> 0.25％Formvar </td><td>電子顕微鏡学</td><td> 15810 </td><td></td></tr><tr><td>警官スロットグリッド当たり</td><td>電子顕微鏡学</td><td> M2010-CR </td><td></td></tr><tr><td> 4％四酸化オスミウム</td><td>電子顕微鏡学</td><td> 19140 </td><td></td></tr><tr><td>抗SOD3抗体</td><td>アブカム</td><td> Ab21974 </td><td></td></tr><tr><td>ヤギ抗ウサギのAlexa Fluor 488 </td><td>インビトロジェン</td><td> A11070 </td><td></td></tr><tr><td>スパーの埋め込み樹脂</td><td>電子顕微鏡学</td><td> 14300 </td><td></td></tr></tbody></table>

mouse eye enucleation for remote high-throughput phenotyping

マウスの眼は人間の眼疾患のトランスレーショナル研究のための重要な遺伝的モデルです。このような黄斑変性、光受容体の変性、白内障、緑内障、網膜芽細胞腫、および糖尿病性網膜症などのヒトでのまばゆいばかりの病気、トランスジェニックマウスで再現されています1月5日ほとんどのトランスジェニックマウスおよびノックアウトマウスは非眼疾患を研究する研究室で生成されましたが、器官系の間の遺伝的保全が同じ遺伝子の多くはまた、眼の発達と疾患における役割を果たしている可能性を示唆している。したがって、これらのマウスは、眼の中に新たな遺伝子型と表現型の相関を発見するための重要なリソースを表します。これらのマウスは世界中に散らばっているので、それは、獲得、維持、効率的で費用対効果の高い方法で表現型、それらをすることは困難である。従って、最も高スループットの眼科表現型画面は生きたマウスで目を調べるために、眼科の専門知識を現場で必要とするいくつかの場所に制限されています6-9は、当研究室が開発した代替アプローチは、トランスジェニックマウスの目の大規模または小規模な調査で使用することができるリモート組織獲得するための方法である。ビデオベースの手術技能移転、組織の固定、出荷のために標準化された手順は、どの研究室では変異動物から全体の目を収集し、分子と形態学的表現型解析のためにそれらを送信することができます。このビデオの記事では、我々は、リモート表現型解析に固定されていないと血流の両方の固定マウスの目を摘出すると、転送する手法を提案する。

Watch this Scientific Journal Video about Mouse Eye Enucleation for Remote High-throughput Phenotyping at JoVE.com

Mouse Eye Enucleation for Remote High-throughput Phenotyping

解剖手法は、ハイスループットスクリーニングで表現型解析を実行するための組織固定のマウス眼球摘出を示しています。

リモートハイスループット表現型解析のためのマウスの目の摘出

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

mouse-eye-enucleation-for-remote-high-throughput-phenotyping

Research

JoVE Journal

Medicine

20.2K ビュー.  University of Iowa. 解剖手法は、ハイスループットスクリーニングで表現型解析を実行するための組織固定のマウス眼球摘出を示しています。

ビデオ: Mouse Eye Enucleation for Remote High-throughput Phenotyping

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

タンパク質治療薬の製造細胞株の開発のためのハイスループット自動化されたプラットフォーム

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

医学

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

血管新生研究のための角膜ポケットアッセイのマウスモデル

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

意識、無拘束マウスにおける高インスリン血症、正常血糖クランプ

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

Remote Magnetic Navigation for Accurate, Real-time Catheter Positioning and Ablation in Cardiac Electrophysiology Procedures

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac substrates such as left atrial flutter. This protocol describes all the clinical and invasive interventional steps performed during a human electrophysiological study and ablation to assess the accuracy, safety and real-time navigation of the Catheter Guidance, Control and Imaging (CGCI) system. Patients who underwent ablation of a right or left atrium flutter substrate were included. Specifically, data from three left atrial flutter and two counterclockwise right atrial flutter procedures are shown in this report. One representative left atrial flutter procedure is shown in the movie. This system is based on eight coil-core electromagnets, which generate a dynamic magnetic field focused on the heart. Remote navigation by rapid changes (msec) in the magnetic field magnitude and a very flexible magnetized catheter allow real-time closed-loop integration and accurate, stable positioning and ablation of the arrhythmogenic substrate.

This report provides a detailed description of a new remote navigation system based on magnetic driven forces, which has been recently introduced as a new robotic tool for human cardiac electrophysiology procedures.

心臓電気生理学プロシージャでの正確なリアルタイムカテーテルポジショニングとアブレーション用のリモート磁気ナビゲーション

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac ...

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

急性移植片対宿主病のバイオマーカーの検証のためのハイスループットシーケンシャルELISA

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

神経保護戦略のための治療因子の送達のために設計成体幹細胞の高スループット特性評価

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

マウス網膜循環における血流動態を調べるための赤血球および白血球の蛍光色素標識

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate (HoLEP).

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the quality of life. BPH includes 2 major treatments: medication and surgery. Surgical treatment is often the most effective and final intervention. Holmium laser enucleation of the prostate (HoLEP), one of the most efficient surgical procedures for BPH, is conducted transurethrally. During surgery, the most important and most difficult part is to locate the surgical capsule of the prostate, which is consistent to the idea of anatomic surgery. Different skills may be needed in treating different parts of the prostate. HoLEP tends to be efficient and safe with good hemostatic properties, and able to treat bladder calculus, which may be the complications of BPH. The technique is particularly suitable for prostate of varying volumes and sizes. Indeed, HoLEP also presents certain disadvantages, such as a long learning curve and costly equipment. Regardless, this method may be the "new standard" and the excellent method for the surgical treatment of BPH.

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate HoLEP.

Here we present a safe and efficient protocol, holmium laser enucleation of the prostate, to treat benign prostatic hyperplasia.

前立腺肥大症の外科治療: ホルミウム レーザー核出術 (HoLEP) 前立腺の。

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

虹彩ルベオーシスのマウス モデルとして虹彩新生血管を穿刺誘発

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.