We thank Lowell Cochran for expert videography assistance. The financial assistance of the Canadian Institutes of Health Research (CIHR), the Canadian Breast Cancer Foundation (CBCF, Ontario Chapter), the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Breast Cancer Research Alliance, the Ontario Centers of Excellence, the Breast Cancer Action Kingston and the Clare Nelson bequest fund through grants to LR is gratefully acknowledged. SG was the recipient of an NSERC studentship. MG was supported by a postdoctoral fellowship from the US Army Breast Cancer Program, the Ministry of Research and Innovation of the Province of Ontario and the Advisory Research Committee of Queen&#8217;s University.

1エレクトロポレーションチャンバー内の細胞をプレーティング<ol><li>層流フード中で、いつものように、滅菌技術を用いて細胞をトリプシン処理。 注：これらは、したがって、ガラス上の細胞のアドヘレンとギャップ結合の形成を妨げる可能性が広がるので、それは、遠心分離によってトリプシンの痕跡をすべて排除することは非常に重要である。 </li><li> 37°Cの CO 2インキュベータ内で、その場でエレクトロ（ 図1）、および場所を提供無菌エレクトロポレーションチャンバー中の細胞懸濁液のピペット1ミリリットル。 注：細胞接着は、フィブロネクチン、コラーゲン、ポリリジン、ま​​たは細胞および組織接着剤（材料表を参照）上にプレーティングすることによって改善することができる。 </li><li>細胞がコンフルエント層を形成しているとき、彼らはGJIC試験の準備ができている。 </li></ol> 2エレクトロポレーション手順 GJIC検査用エレクトロポレーションは、層流フードの外側に行うことができるそれはほんの数分かかりますので、。より長いインキュベーション時間は、特定の実験に必要とされる場合、それは層流フード内で完全に実施することができる。全ての場合において、細胞が増殖されるチャンバは無菌でなければならない。 <ol><li> 5 mg / mlのルシファー黄色の溶液を準備します。2ミリリットルカルシウムを含まない増殖培地中で（エレクトロ付属）10mgのLY粉末を溶かす。より長いインキュベーション時間を必要とする実験のために、4℃。で溶液とストアをフィルター滅菌</li><li>増殖培地を吸引し、単層を傷つけたり、細胞を乾燥しないように注意して、カルシウムを含まない培地で細胞を洗浄。細胞が乾燥している場合は、彼らは通常より暗い核を持っているし、エレクトロポレーションすることなく、LYを拾う。効果はより多くの空気ドラフト（ 図4F）にさらされているスライドの途中でより顕著である。 </li><li>チャンバーの端にある細胞（全体のチャンバのための400μlの）へのエッペンドルフピペット、ピペット色素溶液において、a、bとの細胞層に触れないように注意しeing。 </li><li>エレクトロ付属のホルダーに室を置き、適度な強度（考察を参照）のパルスを適用します。 </li><li>慎重にLY液の一部を吸引。これは、それほど重要での実験のために二度目に再使用することができる。 </li><li> 10％透析血清を含むカルシウムを含まないDMEMを追加します。 </li><li>ギャップ結合を介して色素転写を可能にするためにインキュベーター内で3〜5分間、細胞をインキュベートします。 注：この時点での透析血清を含むことは、気孔の閉鎖を支援します。 </li><li>生細胞観察用のカルシウムを含まないDMEMで取り込まれていない色素を洗ってください。あるいは、細胞をPBS（リン酸緩衝生理食塩水）で洗浄し、その後、ウェルに4％ホルムアルデヒドの添加によって、この段階で固定することができる。すべての場合において、洗浄はすべてのバックグラウンドを削除する必要があります。 注：電圧が低すぎる場合に最適な条件を決定するための予備実験では、それは細胞がincubatoに回復させることが可能である数分間rとスライドのコスト節約するために、同じスライドをもう一度エレクトロポ。 </li></ol> 3顕微鏡検査<ol><li>使用されている染料のための適切なフィルターを装備した倒立顕微鏡を用いて、蛍光照明下で細胞を観察します。黄色ルシファーのために、励起が423nm、放射555nmの、ニコンIX70顕微鏡用WBVフィルタです。 </li><li>位相コントラスト下の細胞形態を調べるために、液体でトップにそれを充填した後、穴プラスチックの上にガラスカバースリップを配置することによって、メニスカス効果を排除します。より良い写真のために、ウェルをスライドから取り外すことができ、カバースリップは、フレーム上に置いた。生きた細胞については、増殖培地で充填されなければならないが、固定した細胞については、ウェルは、顕微鏡観察のためにグリセロールを充填することができる。 注：細胞が色素（上記のステップ2.7）の転送以下のホルムアルデヒドで固定している場合は洗浄して撮影が容易になります。細胞がある場合はありませんtは固定、蛍光はLYの細胞タイプ、電圧、および量に応じて約60分以内に細胞から除去される固定された細胞に蛍光を数時間保持している間に導入した。しかし、蛍光が消えたり、固定した細胞では、O / Nインキュベートした後に消散する。このため、写真は、エレクトロポレーション（ 図2）の後にすぐに注意する必要があります。 </li></ol>細胞間コミュニケーションの4。定量<ol><li>蛍光および位相差（ 図3）の下で20倍を目的とした細胞を撮影。 </li><li>非電気穿孔面積（ 図2、矢印、エレクトロポレーションエッジ）との国境でのエレクトロポレーションした細胞を同定し、スターとマークします。 </li><li>染料はギャップ結合を介して転送されている非電気穿孔側、上の蛍光細胞を特定し、ドットマーク（ 図3Aおよび3B）。 </li><li>の合計数で割り縁部（ 図3Aおよび図3B）に沿ってエレクトロポレーションした細胞の数で非エレクトロポレーション領域上の細胞を蛍光を発する。 注：少なくとも200個の連続電気穿孔境界セルからの転送を各実験について計算される。得られた数は、GJIC値です。 </li></ol>

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The corresponding author is the inventor in a patent held by Queen&#8217;s University, which has been licensed to Cell Projects Inc. The company or the University had no influence upon the contents of the paper.

プロトコルの重要なステップ 電気穿孔材料 エレクトロポレーションされる材料の純度が非常に重要である。細胞は非常に平坦である場合には、追跡用色素のより高い濃度が使用されなければならない（LYための最大20 mg / ml）をそのような場合には、純度がより重要より球形1細胞よりなる。 LY以外に他の染料またはnonpermeant分子の多種多様なその?...

セルへの電流の印加は、エレクトロポレーションと呼ばれるプロセスにより、細胞膜上の細孔の形成を引き起こす。細孔は膜を通してnonpermeant多様な分子の通過を可能にする。形成された細孔が非常に小さく、細胞生理に最小限の妨害で、急速に再閉鎖するように電場が、正確に制御することができる。興味深いことに、接着細胞は懸濁液中でエレクトロポレーションすることが切り離されることなく、それらが成長する表面上にある導電性および透明なインジウム-スズ酸化物（ITO）でコーティングし、 その場で電気穿孔し、スライドガラス上に成長させることができる。細胞は、この表面上に非常によく成長することができ、そしてそれらが結合拡張されると、詳細な顕微鏡観察が可能である。この技術を用いて、小nonpermeant分子は、コンプの活性化に関する研究のためのこの技術は特に適しており、瞬時に細胞の本質的に100％に導入することができる（1件）受容体のリガンド刺激後の経路のonents。 エレクトロポレーション（electrotransfectionも呼ばれる）DNAの導入のために主に使用されてきた。しかしながら、in situでエレクトロポレーションが転写因子の結合、またはsiRNA 3,5阻害するためにアンチセンスRNA、二本鎖DNAデコイオリゴヌクレオチドなどのようなペプチド2-4、オリゴヌクレオチド分子は、多種多様の導入のために有益であることができ、 6、放射性ヌクレオチド7-10、タンパク質11 12またはプロドラッグ13。エレクトロポレーション後、細胞を生化学的分析のために溶解または固定し、抗体で染色することができる。 細胞は、導電性コーティングの縁を横切って成長するように、細胞増殖は、2つの表面の高さの差によって乱されないように導電性ITOコーティングは、800-1,000Å非常に薄い。これは、非electrop利点を提供していますorated細胞を対照として使用するために、エレクトロポレーションのものと並行して成長させることができる。ビデオと同様のアプローチは、ギャップ結合細胞間コミュニケーション（GJIC）の検査のために使用することができる。 ギャップ結合は、隣接するセル14の内部を接続するチャネルである。そのようなsrcと癌遺伝子がGJIC 15,16を抑制しながら、ギャップ結合は、細胞間の通信は、腫瘍形成および転移に重要な役割を果たしている。 GJICを調べるために、イエロールシファー（LY）のような蛍光色素は、多くの場合、マイクロインジェクションを介して培養細胞に導入したりこすっロード17と隣接するセルへの色素の拡散を顕微鏡、蛍光照明下で観察されている。これらの技術は、しかしながら、常に、細胞損傷を引き起こす。現在、細胞が部分的にITO 18で被覆されたガラススライド上で増殖させる技術が記載されている。電気パルスは、CA LY（または他の色素）の存在下で適用した隣接する、非エレクトロポレーションした細胞への染料の移動を顕微鏡、蛍光照明で観察しながら、スライドの導電部に増殖している細胞への浸透を使用して。単層から剥離する傾向が妨害感受性細胞を避けるために、電極を必要としない設計されたアセンブリは、電流19を印加するセルの上に配置される。このアプローチは、多数のセルにGJICを定量する能力を提供し、細胞代謝に検出可能な妨害なしに、血清刺激12を次のG1期の長さに対する効果の欠如によって示されるように、fosのレベルの増加癌原遺伝子タンパク質（ラプティス、未発表）、または細胞ストレス、p38の豚やJNK / SAPKキナーゼ1に関連付けられた2つのキナーゼ。このアプローチは、癌遺伝子発現、形質転換及びGJのレベルの間のリンクの検査を可能にしたIC 18と同様に、肺腫瘍標本20〜23から新たに培養した細胞を含む多様な細胞型におけるGJICの際にはSrcおよびStat3の効果。また、基板への細胞付着はその段階19,24で減少しているが、成功した脂肪細胞分化の際にギャップ結合閉鎖のデモンストレーションのために使用されている上部電極を欠いて説明した設定と、その場でのエレクトロポレーション。

<table border="0" cellpadding="0" cellspacing="0" width="893">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Name of Material/ Equipment</td>
			<td style="width:428px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">DMEM</td>
			<td style="width:428px;">Cellgro http://www.cellgro.com/</td>
			<td style="width:119px;">50-013-PB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">DMEM without Calcium</td>
			<td style="width:428px;">Hyclone (Thermo scientific: http://www.thermoscientific.com)</td>
			<td style="width:119px;">SH30319-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Donor Calf Serum&#160;</td>
			<td style="width:428px;">PAA: http://www.paa.com/</td>
			<td style="width:119px;">Cat.# B15-008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Fetal Bovine Serum</td>
			<td style="width:428px;">PAA: http://www.paa.com/</td>
			<td style="width:119px;">Cat.# A15-751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Electroporation apparatus</td>
			<td style="width:428px;">Cell Projects Ltd UK: http://www.cellprojects.com/</td>
			<td style="width:119px;">ACE-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Chambers</td>
			<td style="width:428px;">Cell Projects Ltd UK</td>
			<td style="width:119px;">ACE-04-CC</td>
			<td>4-wells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Chambers</td>
			<td style="width:428px;">Cell Projects Ltd UK</td>
			<td style="width:119px;">ACE-08-CC</td>
			<td>8-wells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Lucifer Yellow</td>
			<td style="width:428px;">Cell Projects Ltd UK</td>
			<td style="width:119px;">ACE-25-LY</td>
			<td>High purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">CelTak</td>
			<td style="width:428px;">BD Biosciences</td>
			<td style="width:119px;">354240</td>
			<td>Cell and tissue adhesive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Fibronectin</td>
			<td style="width:428px;">Sigma Aldrich</td>
			<td style="width:119px;">F1141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Collagen</td>
			<td style="width:428px;">BD Biosciences</td>
			<td style="width:119px;">354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Poly-Lysine</td>
			<td style="width:428px;">Sigma Aldrich</td>
			<td style="width:119px;">P8920</td>
			<td></td>
		</tr>
	</tbody>
</table>

a functional assay for gap junctional examination; electroporation of adherent cells on indium-tin oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

図2は、固定および洗浄後、ラット肝臓上皮T51Bセル22、LYで電気穿孔し、蛍光（パネルAおよびB）下で撮影、または位相差（C）は、照明を示している。パネルAには、エレクトロポレーション領域のエッジは赤でマークされています。赤線の右側にある蛍光の勾配はギャップ結合を介して転送を示している。染料が転送された細胞は、ドットでマークしながら、エレクトロ?...

Watch this Scientific Journal Video about A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide at JoVE.com

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

ギャップ結合審査のための機能アッセイ;インジウム - スズ酸化物に対する接着した細胞のエレクトロポレーション

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

a-functional-assay-for-gap-junctional-examination-electroporation

Research

JoVE Journal

Biology

9.8K ビュー.  Queen's University. This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

ビデオ: A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues.  In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates.  The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive.  Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule.  Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks.    Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.  Micropatterning of several cell types was demonstrated on these substrates.   In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

光学的に透明な酸化インジウムスズ電極上にパターニングセル

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular ...

生物学

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

MRイメージングと生体内追跡非侵襲のための酸化鉄ナノ粒子で標識ヒトES細胞とhMSCs

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

マウス網膜神経節細胞における遺伝子発現のための子宮内および生体外でのエレクトロポレーションで

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

接着および非接着性の哺乳動物細胞の in situ細胞下分画で

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

異種哺乳動物細胞で発現したイオンチャネルの変異導入と機能解析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

クローン原性アッセイ：付着細胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

3&#39;UTR -レポーターコンストラクトとマイクロRNAを使用してLightSwitchルシフェラーゼアッセイシステムによるヒトマイクロRNAの識別対象は付着細胞へのミミック

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

の検討<em&gt;ショウジョウバエ</em光学顕微鏡で&gt;幼虫気管ターミナルセル

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

外酸生産の測定と解析は、解糖速度を決定するために

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

ゼブラフィッシュにおける腎毒素マイクロインジェクションは急性腎障害をモデルに

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...