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.细胞培养注意：所有的细胞培养协议必须使用适当的细胞培养技术来执行（参考见细胞生物学的第3章：实验室手册，第I卷10）。 HEI-OC1细胞不要求的适当附着和生长的细胞培养皿的任何附加涂层或处理。非常重要：不要用细胞培养的目的玻璃器皿菜肴;表型和细胞对药物的药物的生物反应会改变（G Kalinec＆F Kalinec，未发表）;建议使用传统的塑料细胞培养皿（见材料/设...

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在本报告中，我们描述了如何培养开平OC1细胞，并利用它们来评价药物诱导的细胞毒性的机制，并调查prestin无，耳蜗外毛细胞的分子马达的功能特性。该技术程序，然而，有足够一般可以很容易地适应不同的研究。 这里描述的所有协议需要正确的使用成熟细胞培养技术10。就像任何其他细胞系，以开平OC1细胞工作，需要一个适当装备的细胞培养设备，具有认证生物安...

科尔蒂1的房子耳研究所-器官（HEI-OC1）细胞是从转基因小鼠1,2的听觉器官的。在33℃/ 10％CO 2（在允许的条件）从此转基因小鼠的任何细胞的温育诱导触发去分化和加速扩散的永生化基因的表达;移动单元至39℃/ 5％CO 2（非许可条件）导致减少的增殖，分化和，至少在开平OC1，细胞死亡2,3的情况。 HEI-OC1细胞克隆和表征在我们的实验室在十多年前，和最初的研究表明，它们表达耳蜗毛细胞，如prestin无，肌球蛋白7a中，Atoh1的，BDNF钙结合蛋白和钙调蛋白的特异性标志物，而且还支持标记细胞如连接蛋白26和成纤维细胞生长因子受体（FGF-R）2。因此，有人建议，HEI-OC1可以表示的commoñ祖为2尔蒂的器官感觉和支持细胞。平行研究提供了有力的证据表明，典型的耳毒性药物如顺铂，庆大霉素，链霉素诱导的caspase-3的活化在这些细胞中，而药物视为非耳毒性，如青霉素，没有2,3。因此，该细胞系被提出作为在体外系统来研究参与耳毒性及潜在耳毒性或新的药理学药物otoprotective性质筛选的细胞和分子机制。据估计，HEI-OC1细胞已经发表在过去十年超过150研究中使用。 而在寻找不同的药物的潜在促凋亡作用是最涉及该细胞系研究 ​​的主要目标，其他重要的细胞过程像自噬和衰老刚刚开始HEI-OC1细胞4-7进行调查。一世呐最近我们实验室8研究中，我们采用HEI-OC1细胞收集一套完整的有关细胞死亡，存活，增殖，衰老和自噬在临床上经常使用不同的药理学药物诱发的数据。我们还比较了一些开平OC1细胞与那些从HEK-293（人胚肾细胞）和HeLa（人上皮细胞）接受相同治疗的响应。我们的结果表明，开平OC1细胞的每种药物响应的特征的方式，具有鲜明的剂量和时间依赖的灵敏度所研究的机制中的至少一个。我们还强调，在这项研究的实验结果的正确解释，需要与多个技术进行8平行研究。 在不同的研究中，我们调查了采用HEI-OC1细胞来评价prestin无的功能反应，耳蜗外毛细胞马达蛋白（外毛细胞）9 </s了&gt;。我们报道流式细胞仪和激光扫描共聚焦显微镜研究上prestin无表达在容许（P-HEI-OC1）和非培养的HEI-OC1细胞的模式，以及非线性电容（NLC）和全细胞膜片钳研究容许（NP-HEI-OC 1）的条件。我们的结果表明，在NP-HEI-OC 1细胞以时间依赖性的方式既总prestin无表达和血浆膜定位增加。有趣的是，我们还发现，在含有的Na + K + ATP酶的减少，这从质膜易位到细胞质而不在总细胞表达显著变化相关的NP-HEI-OC1细胞的质膜的增加prestin无定位。此外，我们表明，P-HEI-OC 1细胞具有强大的NLC关联prestin无运动功能，当存在于质膜prestin无分子的密度增加，这降低。总之，这些结果强烈支持HEI-OC的用处1细胞研究听觉蛋白质。 在这个视频文章我们将描述如何培养开平OC1细胞，为什么它是方便使用的细胞在允许的条件（P-HEI-OC 1）细胞毒性的研究越来越大，如何评估药物诱导的细胞毒性，以及如何的机制/ s的执行电生理学研究（ 如 ，膜片钳，非线性电容（NLC））调查prestin无，耳蜗外毛细胞的分子马达的功能特性。

<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.

在一对夫妇最近的出版物中，我们报道了一整套旨在评估HEI-OC1细胞对几种常用药理学药物的反应，以及调查prestin无功能8,9研究。在这些研究中，我们利用了在前面部分中描述的所有协议。 一这些以前的研究的结果是，开平OC1细胞在非允许条件培养（39℃/ 10％的CO 2 = NP-HEI-OC 1）显示出在细胞活力高度显著下降?...

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.

与听觉HEI-OC1细胞工作

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 ...

The overall goal of this protocol is to describe the basic procedures to work with HEI-OC1 cells, which can be used to study the effects of pharmacological drugs on auditory cells, and assist in the functional characterization of the motor protein prestin. HEI-OC1 cells are used by many researchers around the world in different experiments with different goals and using different techniques. Hopefully, the procedures described here will provide a common base to make these studies comparable.Of course, these experimental procedures may be further improved. But the differences with those described here could be key to understand and explain possibly contradictory results. In this demonstration we will describe how to work with HEI-OC1 cells starting from a frozen aliquot to having samples ready to perform different studies with widely available techniques.To begin this procedure remove a vial of cryopreserved HEI-OC1 cells from liquid nitrogen and place it in a water bath at 37 degrees Celsius. Submerge only the lower half of the vial and allow it to thaw until only a small amount of ice remains in the vial. In a biological safety cabinet, wipe the outside of the vial with 70%alcohol.Then, pipette the cells from the vial and deliver the entire volume drop by drop, slowly, into a 15 milliliter conical tube containing 10 milliliters of prewarmed growth medium such as DMEM supplemented with 10%FBS. Next, remove the DMSO by centrifuging the tube for five minutes. After five minutes, discard the supernatant and resuspend the cells in nine milliliters of fresh growth medium.Disaggregate the clumps or sheets of cells by fluxing and refluxing the suspended cells between the pipette and the medium three to four times with the tip of the pipette touching the bottom of the tube. Then, place the total volume of cells suspended in growth medium in a 100 millimeter diameter untreated plastic cell culture dish. Incubate the cells at 33 degrees Celsius with 10%CO2.In this step, remove the old medium and wash the cells with two milliliters of PBS. Then, cover the cell monolayer with 0.25%trypsin. Examine the cells using an inverted microscope when 40%of them have been detached.If necessary, gently rock the culture dish to release any remaining attached cells Resuspend the cells and remove the trypsin by centrifuging the tube for five minutes. Afterward, discard the supernatant and resuspend the cells in nine milliliters of fresh growth medium. Next, disaggregate the clumps or sheets of cells by fluxing and refluxing the suspended cells between the pipette and the medium, three to four times with the tip of the pipette touching the bottom of the tube.Proceed to seed the cells in 100 millimeter diameter cell culture dishes. Incubate them at 33 degrees Celsius with 10%CO2 and divide them as many times as necessary for the planned experiments, or for generating a new stock. For differentiation, incubate HEI-OC1 cells at the permissive conditions until they reach about 80%confluence.Then, incubate the cells at 39 degrees Celsius with 5%CO2 for two to three weeks and change the medium every other day to remove dead cells. Begin this procedure by counting the cells using either an automatic cell counter or a hemocytometer and adjust the cell concentration to the required value. Next, seed the cells in a 96 well clear flat bottom plate, a white walled 96 well plate, or a six well plate.If required by the particular assay, incubate the cells overnight for substrate attachment before treating them with the drugs of interest. Then, measure the outcome of the treatment by flow cytometry or another adequate technique. To harvest the cells, wash them twice with 10 milliliters of calcium-and magnesium-free PBS.Add two milliliters of the enzyme-free detacher solution and incubate the cells for three minutes at 37 degrees Celsius with 5%CO2. Use an inverted microscope to check the detachment of the cells. If necessary, move the cell culture dish gently to detach more cells, and be careful not to shake it.Next, add 10 milliliters of DMEM plus 10%FBS and pipette the cells up and down, gently, five times. After that, look at the cells under a microscope. If more than 80%of the cells are already separated, no further pipetting is needed.If the cells are still in clusters, continue gently pipetting until more than 80%of the cells are no longer in clusters. An optical control of the cells should show many single round cells with smooth membrane edges. Afterwards, proceed with patch-clamp and NLC measurements, as described in the text protocol.HEI-OC1 cells cultured at nonpermissive conditions showed a highly significant decrease in cell viability, and an equally significant increase in cell death in respect to cells cultures at permissive conditions. Since drug effects on cells already dying or with a decreased viability could be different than the effects of the same drug on healthy cells, considerable efforts should be dedicated to discriminate drug effects from the effects of culture conditions in any cytotoxicity study performed on NP-HEI-OC1 cells. Therefore, we recommend using HEI-OC1 cells cultured at permissive conditions for experiments that do not specifically require differentiated cells.The confocal images shown here confirm the expression of the auditory motor protein prestin in HEI-OC1 cells cultured at 33 degrees Celsius and 39 degrees Celsius. Note that prestin immunolocalizes mostly at the cytoplasm of HEI-OC1 cells growing in the permissive conditions, and it is more concentrated at the plasma membrane in the more differentiated cells. Flow cytometry studies showed a clear increase in prestin expression in both conditions, suggesting that translocation was accompanied by synthesis of more prestin molecules.On the other hand, the electrophysiological studies showed a robust NLC in HEI-OC1 cells growing at 33 degrees Celsius, with a peak value that decreased and a voltage at peak capacitance that shifted to the more depolarized values as the cells were moved to the nonpermissive conditions for one and two weeks. Intriguingly, these results suggest that motor function could be compromised if the density of prestin molecules in the plasma membrane is too high. It is important to remember that the protocols described here require the correct use of well-established cell culture techniques.Also, remember to avoid the use of antibiotics. In addition, remember that the phenotype and response of HEI-OC1 cells will strongly depend on the culture parameters.

Research

JoVE Journal

Biology

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

与非侵入性检测荧光染料标记的干细胞与光学成像

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 ...

科研

生物学

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

映射后的Theta突发刺激对人类听觉皮层功能成像

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

诱导上皮和分析间质转化

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

荧光成像与一纳米精度（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

转染RAW264.7细胞用荧光素酶报告基因

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

灭鼠工作心脏模型心肌的性能和氧耗的研究

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

肾毒素显微注射斑马鱼到模型急性肾损伤

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

为葡萄糖和脂肪酸氧化率在大鼠离体的测定方法

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

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

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

细菌、酵母和人类细胞中瞬态形成的分子茶龙组件的原位监测

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 ...