Name: working with auditory hei-oc1 cells
Uploaded: 2016-09-03T14:00:00
Description: Watch this Scientific Journal Video about Working with Auditory HEI-OC1 Cells at JoVE.com

This work was supported by NIH Grants R01-DC010146 and R01-DC010397. Its content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health.

1.細胞培養注：すべての細胞培養プロトコルは、適切な細胞培養技術を使用して実行されなければならない（：実験室ハンドブックVolume I 10参照用細胞生物学の最初の3章を参照のこと）。 HEI-OC1細胞は、適切な接着および増殖のための細胞培養皿の任意の追加のコーティングまたは治療を必要としません。非常に重要：細胞培養​​の目的のためにガラス製品食器?...

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このレポートでは、どのように文化HEI-OC1細胞をに説明し、薬剤誘発性細胞毒性のメカニズムを評価するためにそれらを使用し、プレスチン、蝸牛OHCSの分子モーターの機能特性を調査します。技術的手順は、しかしながら、容易に別の試験に適合することが十分に一般的です。 ここで説明するすべてのプロトコルは、十分に確立された細胞培養技術10の正しい使?...

コルチ1のハウス耳研究所-臓器（HEI-OC1）細胞は、トランスジェニックマウスの1,2の聴覚器官に由来しています。 33℃/ 10％CO 2（許容条件）で、このトランスジェニックマウスからの任意の細胞のインキュベーションは、脱分化と加速増殖をトリガーする不死化遺伝子の発現を誘導します。 39℃/ 5％CO 2の少なくともHEI-OC1、細胞死2,3の場合には、増殖、分化を減少した（非許容条件）リードに対して細胞を移動させます。 HEI-OC1細胞をクローニングし、十年以上前に私たちの研究室で特徴付けられ、そして初期の研究は、彼らが特定のそのようなプレスチン、ミオシン7aと、のAtoh1、BDNF、カルビンジンおよびカルモジュリンなどの蝸牛有毛細胞のマーカーだけでなく、支援のマーカーを発現することが示されましたコネキシン26及び線維芽細胞増殖因子受容体（FGF-R）2のような細胞です。したがって、それはHEI-OC1はのCommoを表すことが示唆されましたコルチ2の器官の感覚と支持細胞のためのn個の前駆細胞。薬は、非耳毒性考えながら並列研究はペニシリンのように、2,3なかった、シスプラチン、ゲンタマイシンおよびストレプトマイシンのような典型的な耳毒性薬が誘発されることをこれらの細胞におけるカスパーゼ3活性化を強力な証拠を提供しました。従って、この細胞株は、耳毒性に関与する細胞および分子機構を調査し、新しい薬理学的薬剤の潜在的な耳毒性またはotoprotective特性のスクリーニングするためのインビトロ系として提案されました。 HEI-OC1細胞を、過去10年間に発表された百以上及び五十の研究において使用されていると推定されます。 異なる薬剤の潜在的なプロアポトーシス効果を見ているが、この細胞株を含む研究のほとんどの主要な目標であったが、オートファジーと老化のような他の重要な細胞プロセスはちょうどHEI-OC1細胞4-7で調査され始めています。私naが我々の研究室8からの最近の研究では、我々は頻繁に診療所で使用される異なる薬理学的薬剤によって誘導される細胞死、生存、増殖、老化およびオートファジーに関するデータの包括的なセットを収集するためにHEI-OC1の細胞を使用しました。我々はまた、同一の治療を受けてHEK-293（ヒト胎児腎臓細胞）およびHeLa細胞由来のもの（ヒト上皮細胞）でHEI-OC1細胞の応答の一部を比較しました。我々の結果は、HEI-OC1細胞を、試験下のメカニズムの少なくとも1つに特有の用量及び時間依存性の感度で、特性ようにザ各薬物に応答することが示されました。また、実験結果の正しい解釈は、複数の技術8と平行した研究を行う必要がありますことをその研究に強調しました。 別の研究では、プレスチンの機能的応答を評価するためのHEI-OC1細胞の使用、蝸牛外有毛細胞のモータータンパク質（OHCS）9を調査しました</s&gt;アップ。私たちは、許容（P-HEI-OC1）および非で培養HEI-OC1細胞におけるプレスチン発現パターンと同様に、非線形容量（NLC）および全細胞パッチクランプ研究にフローサイトメトリーおよび共焦点レーザー走査顕微鏡研究報告しました許容（NP-HEI-OC1）条件。我々の結果は、NP-HEI-OC1細胞で時間依存的に総プレスチン発現および原形質膜局在化の増加の両方。興味深いことに、我々はまた、全細胞発現の有意な変化なしに細胞質への形質膜からトランスロケーションのNa + K + ATPアーゼの減少と相関NP-HEI-OC1の細胞の細胞膜でのプレスチンの局在の増加ことがわかりました。また、我々は、P-HEI-OC1細胞は原形質膜に存在するプレスチン分子の密度が増加したときに減少し、運動機能を、プレスチンに関連付け堅牢なNLCを有することを実証しました。要するに、これらの結果は、HEI-OCの有用性をサポート1細胞は、聴覚タンパク質を調査します。 このビデオの記事では、我々は文化HEI-OC1細胞に、なぜ薬剤誘発性細胞毒性のメカニズム/秒を評価する方法、細胞毒性研究のために許容条件（P-HEI-OC1）で増殖する細胞を使用するのが便利であるとどのように方法について説明します電気生理学的研究を行うために（ 例えば 、パッチクランプ、非線形キャパシタンス（NLC））はプレスチン、蝸牛OHCSの分子モーターの機能的特性を調べました。

<table border="0" cellpadding="0" cellspacing="0" width="1447">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:905px;">HEI-OC1 cells</td>
			<td style="width:143px;"></td>
			<td style="width:139px;"></td>
			<td style="width:260px;">ALL THE ASSAY KITS, EQUIPMENTS&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Class II Biological Safety cabinet</td>
			<td>The Baker Company</td>
			<td>Sterilgard III</td>
			<td>AND COMPANIES INDICATED IN THE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Refrigerated centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>PREVIOUS 2 COLUMNS ARE ONLY</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted microscope</td>
			<td>Zeiss</td>
			<td>Axiovert 25</td>
			<td>EXAMPLES, AND ANY OTHER SIMILAR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Waterbath</td>
			<td>Stovall</td>
			<td>HWB115</td>
			<td>PRODUCT COULD BE USED.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell counter</td>
			<td>Nexcelom</td>
			<td>Cellometer Auto T4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Two (2) Cell incubators, one at 33&#176;C/10% CO2 and other at 39&#176;C/5% CO2</td>
			<td>Forma Scientific</td>
			<td>3110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture dishes, PS, 100 x 20 mm with vents</td>
			<td>Greinier Bio-One</td>
			<td>664-160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture dishes , PS,&#160; 60 x 15 mm with vents&#160;</td>
			<td>Greiner Bio-One</td>
			<td>628160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellstar tissue culture flasks&#160; 250 mL</td>
			<td>Greiner Bio-One</td>
			<td>658-175</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellstar tissue cultur&#160; flasks 550 mL</td>
			<td>Greiner Bio-One</td>
			<td>660-175</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;6 well cell culture plate, with lid-Cellstar</td>
			<td>Greiner Bio-One</td>
			<td>657-160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microtest Tissue culture plate, 96 well,flat bottom with lid</td>
			<td>Becton Dickinson</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-Assay-Plate, Chimmey, 96-well white,clear botton</td>
			<td>Greiner Bio-One</td>
			<td>655098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml Polypropylene conical tube with cap Cellstars</td>
			<td>Becton Dickinson&#160;</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 ml Polypropylene conical tubes with cap-Cellstars</td>
			<td>Greiner Bio-One</td>
			<td>188-271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS pH 7.4 (1X)&#160;</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td>Life Technologies</td>
			<td>11965-084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)&#160;</td>
			<td>Hyclone</td>
			<td>SH10073.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leibovitz&#39;s L-15 Medium, no phenol red</td>
			<td>Gibco/Invitrogen</td>
			<td>21083-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin, 0.25%&#160;</td>
			<td>Life Technologies</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TACS MTT Cell Proliferation Assay Kit</td>
			<td>Trevigen</td>
			<td>4890-25-K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Caspase-Glo 3/7 Assay&#160; kit</td>
			<td>Promega</td>
			<td style="width:139px;">&#160; G8091&#160;</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BrdU Cell Proliferation Assay Kit&#160;</td>
			<td>Cell Signaling</td>
			<td>6813</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-enzymatic cell dissociation solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C5789</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell-Tox Green Cytotoxicity Assay Kit</td>
			<td>Promega</td>
			<td style="width:139px;">&#160; G8741&#160;</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACSAriaIII instrument&#160;</td>
			<td>BD Biosciences</td>
			<td>&#160;FACSAriaIII</td>
			<td>With 488 nm excitation (blue laser)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital Blot Scanner</td>
			<td>LI-COR</td>
			<td>C-DiGit</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electrophoresis and Blotting Unit</td>
			<td>Hoefer</td>
			<td>SE300 miniVE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectra Max 5 Plate Reader with Soft Max Pro 5.2 Software</td>
			<td>Molecular Devices</td>
			<td>SpectraMax 5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Patch-clamp amplifier</td>
			<td>HEKA</td>
			<td>EPC-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puller for preparing patch electrodes</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td></td>
		</tr>
	</tbody>
</table>

working with auditory hei-oc1 cells

HEI-OC1 is one of the few mouse auditory cell lines available for research purposes. Originally proposed as an in vitro system for screening of ototoxic drugs, these cells have been used to investigate drug-activated apoptotic pathways, autophagy, senescence, mechanism of cell protection, inflammatory responses, cell differentiation, genetic and epigenetic effects of pharmacological drugs, effects of hypoxia, oxidative and endoplasmic reticulum stress, and expression of molecular channels and receptors. Among other several important markers of cochlear hair cells, HEI-OC1 cells endogenously express prestin, the paradigmatic motor protein of outer hair cells. Thus, they can be very useful to elucidate novel functional aspects of this important auditory protein. HEI-OC1 cells are very robust, and their culture usually does not present big complications. However, they require some special conditions such as avoiding the use of common anti-bacterial cocktails containing streptomycin or other antibiotics as well as incubation at 33 &#176;C to stimulate cell proliferation and incubation at 39 &#176;C to trigger cell differentiation. Here, we describe how to culture HEI-OC1 cells and how to use them in some typical assays, such as cell proliferation, viability, death, autophagy and senescence, as well as how to perform patch-clamp and non-linear capacitance measurements.

最近の出版物のカップルで、私たちはいくつかの一般的に使用される薬理学的薬剤へのHEI-OC1細胞の応答を評価するだけでなく、プレスチン機能8,9を調査することを目的とした研究の包括的なセットを報告しました。これらの研究において、我々は、前のセクションで説明したすべてのプロトコルを利用しました。 <p class="jove_content" fo:keep-together.within-page="1"...

Watch this Scientific Journal Video about Working with Auditory HEI-OC1 Cells at JoVE.com

Working with Auditory HEI-OC1 Cells

House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few mouse auditory cell lines currently available for research purposes. This protocol describes how to work with HEI-OC1 cells to investigate the cytotoxic effects of pharmacological drugs as well as functional properties of inner ear proteins.

聴覚HEI-OC1細胞を使用した作業

HEI-OC1 is one of the few mouse auditory cell lines available for research purposes. Originally proposed as an in vitro system for screening of ototoxic ...

Research

JoVE Journal

Biology

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

光イメージングによる非侵襲的検出のための蛍光色素で標識幹細胞

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

機能イメージングによるヒト聴覚皮質上のシータバースト刺激の後遺症のマッピング

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Induction and Analysis of Epithelial to Mesenchymal Transition

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence Imaging with One-nanometer Accuracy FIONA

ワンナノメートルの精度で蛍光イメージング（FIONA）

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

ルシフェラーゼレポーター遺伝子でRAW264.7細胞をトランスフェクト

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

心筋パフォーマンスと酸素消費量の研究のための齧歯類のワーキングハートモデル

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

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 ...

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

単離されたワーキングラット心臓におけるグルコースおよび脂肪酸酸化の宿泊料金を決意するための方法

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

動的細胞突起におけるソフトウェア支援トラッキングのためのグラフィカルユーザインタフェース

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

細菌、酵母、ヒト細胞における一過性形成分子シャペロンアセンブリのモニタリング

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...