Name: in vivo-like organotypic murine retinal wholemount culture
Uploaded: 2010-01-11T17:30:00
Description: Watch this Scientific Journal Video about In vivo-like Organotypic Murine Retinal Wholemount Culture at JoVE.com

著者は、技術支援のための器官培養およびU. LaubとU.ゲルスターの設立と初期支援のためにE.デラロサとAI Valencianoに感謝します。

すべての機器および試薬は、滅菌購入する必要がありますまたは熱または蒸気滅菌または70％エタノールで滅菌する必要があります。 動物実験は動物実験で定められている実験的な手続きと規制のための動物の管理と使用に関するNI​​Hのガイドラインに従って、欧州共同体理事会指令（86/609/EEC）に準拠して行われたという著者らの状態ケアとデュースブルク - エッセ?...

<ol>
	<li>Rieke, M., Gottwald, E., Weibezahn, K. -. F., Layer, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+reconstruction+in+3D-spheroids+from+rodent+retina+in+a+motion-free,+bioreactor-based+microstructure.">Tissue reconstruction in 3D-spheroids from rodent retina in a motion-free, bioreactor-based microstructure.</a> Lab. Chip. 8, 2006-2213 (2008).</li><li>Donovan, S. L., Dyer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+square+wave+electroporation+of+retinal+explant+cultures.">Preparation and square wave electroporation of retinal explant cultures.</a> Nature Protocols. 1, 2710-2718 (2006).</li><li>Duenker, N., Valenciano, A. I., Franke, A., Hernandez-Sanchez, C., Dressel, R., Behrendt, M., de Pablo, F., Krieglstein, K., de la Rosa, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balance+of+pro-apoptotic+transforming+growth+factor-beta+and+anti-apoptotic+insulin+effects+in+the+control+of+cell+death+in+the+postnatal+mouse+retina.">Balance of pro-apoptotic transforming growth factor-beta and anti-apoptotic insulin effects in the control of cell death in the postnatal mouse retina.</a> Eur. J. Neurosci. 22, 28-38 (2005).</li><li>Franke, A. G., Gubbe, C., Beier, M., Duenker, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factors+beta+and+Bone+morphogenetic+proteins:+Cooperative+players+in+chick+and+murine+programmed+retinal+cell+death.">Transforming growth factors beta and Bone morphogenetic proteins: Cooperative players in chick and murine programmed retinal cell death.</a> J. Comp. Neurol. 495, 263-278 (2005).</li><li>de la Rosa, E. J., D&#237;az, B., De Pablo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoculture+of+the+chick+embryonic+neuroretina.">Organoculture of the chick embryonic neuroretina.</a> Curr. Top. Dev. Biol. 36, 133-144 (1998).</li><li>Dohle, D. S., Pasa, S. D., Gustmann, S., Laub, M., Wissler, J. H., Jennissen, H. P., Duenker, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+ex+ovo+culture+and+ex+ovo+CAM+assay:+How+it+really+works.">Chick ex ovo culture and ex ovo CAM assay: How it really works.</a> J Vis Exp. 32, (2009).</li></ol>

解離、単分子層以上のマウス器官網膜wholemount文化2-5、retinospheroidまたは再集計された3D回転楕円体の培養神経細胞の相互作用との接続の保全1嘘、の利点は 、in vivo の状況で模倣した。旧レポート2〜比較して、私たちのビデオの記事では、網膜を損傷することなく、マウスの目とレンズと硝子体の除去を含む、さまざまな発育段階の網膜の解剖摘出...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Mice</td>
<td>Animal</td>
<td>Charles Rivers Laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dissection microscope</td>
<td>Tool</td>
<td>ZEISS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>Reagent</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>PBS should be cold (&gt; 4&deg;C) and sterile</td>
</tr>
<tr>
<td>Dulbecco`s modified eagle`s medium / nutrient mixture F-12 Ham</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D 8900</td>
<td>DMEM / F-12</td>
</tr>
<tr>
<td>Apo-transferin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T 1147</td>
<td>&nbsp;</td>
<tr>
<td>Putrescin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P 5780</td>
<td>&nbsp;</td>
<tr>
<td>Sodium selenite</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S 9133</td>
<td>&nbsp;</td>
<tr>
<td>Progesterone</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P 6149</td>
<td>&nbsp;</td>
<tr>
<td>Gentamicine</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>25030-024</td>
<td>200 mM (100X), liquid</td>
</tr>
<tr>
<td>Bovine serum albumine (BSA)</td>
<td>Reagent</td>
<td>Roth</td>
<td>8076.3</td>
<td>30 mg/ml</td>
</tr>
<tr>
<td>Collagenase</td>
<td>Reagent</td>
<td>Sigma</td>
<td>C 0773</td>
<td>200 U/ml</td>
</tr>
<tr>
<td>Trypsin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T4799</td>
<td>From porcine pancreas; 1 mg/ml</td>
</tr>
<tr>
<td>Hyaluronidase</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H 3884</td>
<td>200 mg/ml</td>
</tr>
<tr>
<td>DNase I</td>
<td>Reagent</td>
<td>Roche</td>
<td>1 284 932</td>
<td>10 mg/ml</td>
</tr>
<tr>
<td>EDTA</td>
<td>Reagent</td>
<td>Sigma</td>
<td>E 6511</td>
<td>&nbsp;</td>
<tr>
<td>Silicone solution</td>
<td>Reagent</td>
<td>Serva</td>
<td>35130</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde (PFA)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P6148</td>
<td>8% PFA in 0.1M phosphate buffer (pH 7.4).</td>
</tr>
<tr>
<td>4',6-diamidino-2-phenylindole dihydrochloride</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D 0542</td>
<td>DAPI</td>
</tr>
<tr>
<td>Fluorescent Mounting Medium</td>
<td>Reagent</td>
<td>Dako</td>
<td>S3023</td>
<td>&nbsp;</td>
<tr>
<td>BrDU</td>
<td>Reagent</td>
<td>Sigma</td>
<td>B 9285</td>
<td>&nbsp;</td>
<tr>
<td>96-well plates</td>
<td>Tool</td>
<td>FALCON</td>
<td>3072</td>
<td>&nbsp;</td>
<tr>
<td>24-well plates</td>
<td>Tool</td>
<td>FALCON</td>
<td>3047</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur pipettes</td>
<td>Tool</td>
<td>Brand</td>
<td>747720</td>
<td>&nbsp;</td>
<tr>
<td>Forceps DUMONT #5</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11252-30</td>
<td>bevelled very fine shanks (0.05 mm x 0.02 mm tip)</td>
</tr>
<tr>
<td>Forceps DUMONT #7</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11271-30</td>
<td>curved shanks (0.07 mm x 0.10 mm tip)</td>
</tr>
<tr>
<td>Spring scissors,straight, 8cm</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>15000-00</td>
<td>fine, small straight blades</td>
</tr>
<tr>
<td>Standard scissors, straight, sharp/blunt </td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14007-14</td>
<td>Use for decapitation or cervical dislocation</td>
</tr>
<tr>
<td>Eppendorf tubes</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>2ml; round bottom for better precipitation of pellet during centrifugation /cytospin</td>
</tr>
<tr>
<td>Cooling centrifuge</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rotation shaker</td>
<td>Tool</td>
<td>CAT</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cytospin</td>
<td>Tool</td>
<td>Thermo Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
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in vivo-like organotypic murine retinal wholemount culture

動物モデルの遺伝子と分析の対象とアブレーションは、入学する特定の網膜遺伝子機能のための古典的な戦略です。しかしながら、トランスジェニックは、網膜特異的またはコンディショナルノックアウトマウスモデルは、しばしば早期致死を表示したり、胎児や出生後早期の段階を超えて解析を防止する、深刻な奇形に苦しんでいます。 初代培養細胞は、外因的に適用される組換え因子、制御された環境での遺伝子やsiRNAによる遺伝子ノックダウンの過剰発現の影響を調べるための代替です。解離細胞の培養は、それによって薬理学的操作の後に外生的にトリガーの効果の識別を容易に、標的細胞に到達する内因性信号が低下するという利点があります。しかし、重要な細胞間相互作用は、最初に再集計されたretinospheroid文化1が使用されている場合でも、酵素消化または機械的な解離によって破壊されています。 これとは対照的に、器官型網膜wholemount文化が神経細胞の相互作用との接続はまだ保持さ2〜5で生体状況に近い生理的にシステムを提供しています。 このビデオの記事では、胚、生後および成体マウスの目の解剖の特殊性と神経の分析のため（2）解離とサイトスピン手順を含む器官網膜wholemountの文化のようなin vivoでの（1）設立のステップのデモンストレーションでステップを提供するアポトーシスと外因的に適用される組換え因子の存在下で培養した後、例えば器官wholemountsにおける網膜細胞の増殖、。

Watch this Scientific Journal Video about In vivo-like Organotypic Murine Retinal Wholemount Culture at JoVE.com

In vivo-like Organotypic Murine Retinal Wholemount Culture

このビデオの記事では、器官型網膜wholemount文化や外因性誘導効果の分析のためのサイトスピン手順の確立を示しています。器官型網膜wholemount文化模倣体<em&gt; in vivoで</em&gt;状況と古典的なマウスの動物モデルの欠点を回避しながら大幅に実験操作のためのマウス網膜のアクセシビリティを促進する。

生体のような器官型マウス網膜Wholemountの文化で

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

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Biology

In vivo-like Organotypic Murine Retinal Wholemount Culture

Organotypic Culture of Adult Rabbit Retina

大人のウサギの網膜の器官培養

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

Organotypic Slice Culture of E18 Rat Brains

E18ラット脳の器官型スライス培養

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

光の全細胞録音は網膜スライスにおける興奮性シナプス電流を誘発

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

全反射顕微鏡による網膜双極細胞におけるイメージングのエキソサイトーシス

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

初代培養およびコルチのマウス臓器のプラスミドエレクトロ。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

マウス造血幹細胞およびリネージュコミット前駆細胞の表現型の解析と分離

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Isolation and Primary Culture of Rat Hepatic Cells

ラット肝細胞の単離及び初代培養

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

イメージングと免疫組織化学のための網膜ホールマウントの準備のために最適化されたプロトコル

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

ヒトiPS細胞のマイクロRNA発現プロファイル、IPから派生網膜色素上皮、および胎児の網膜色素上皮

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

細胞外のフラックスを用いた Explanted 網膜組織のエネルギー代謝の測定

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...