与听觉HEI-OC1细胞工作
Summary
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.
Abstract
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 °C to stimulate cell proliferation and incubation at 39 °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.
Introduction
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>。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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| HEI-OC1 cells | ALL THE ASSAY KITS, EQUIPMENTS | ||
| Class II Biological Safety cabinet | The Baker Company | Sterilgard III | AND COMPANIES INDICATED IN THE |
| Refrigerated centrifuge | Eppendorf | 5810R | PREVIOUS 2 COLUMNS ARE ONLY |
| Inverted microscope | Zeiss | Axiovert 25 | EXAMPLES, AND ANY OTHER SIMILAR |
| Waterbath | Stovall | HWB115 | PRODUCT COULD BE USED. |
| Cell counter | Nexcelom | Cellometer Auto T4 | |
| Two (2) Cell incubators, one at 33°C/10% CO2 and other at 39°C/5% CO2 | Forma Scientific | 3110 | |
| Cell culture dishes, PS, 100 x 20 mm with vents | Greinier Bio-One | 664-160 | |
| Cell culture dishes , PS, 60 x 15 mm with vents | Greiner Bio-One | 628160 | |
| Cellstar tissue culture flasks 250 mL | Greiner Bio-One | 658-175 | |
| Cellstar tissue cultur flasks 550 mL | Greiner Bio-One | 660-175 | |
| 6 well cell culture plate, with lid-Cellstar | Greiner Bio-One | 657-160 | |
| Microtest Tissue culture plate, 96 well,flat bottom with lid | Becton Dickinson | 353072 | |
| Micro-Assay-Plate, Chimmey, 96-well white,clear botton | Greiner Bio-One | 655098 | |
| 50 ml Polypropylene conical tube with cap Cellstars | Becton Dickinson | 352070 | |
| 15 ml Polypropylene conical tubes with cap-Cellstars | Greiner Bio-One | 188-271 | |
| PBS pH 7.4 (1X) | Life Technologies | 10010-023 | |
| Dulbecco’s Modified Eagle’s Medium (DMEM) | Life Technologies | 11965-084 | |
| Fetal bovine serum (FBS) | Hyclone | SH10073.1 | |
| Leibovitz's L-15 Medium, no phenol red | Gibco/Invitrogen | 21083-027 | |
| Trypsin, 0.25% | Life Technologies | 25200-056 | |
| TACS MTT Cell Proliferation Assay Kit | Trevigen | 4890-25-K | |
| Caspase-Glo 3/7 Assay kit | Promega | G8091 | |
| BrdU Cell Proliferation Assay Kit | Cell Signaling | 6813 | |
| Non-enzymatic cell dissociation solution | Sigma-Aldrich | C5789 | |
| Cell-Tox Green Cytotoxicity Assay Kit | Promega | G8741 | |
| FACSAriaIII instrument | BD Biosciences | FACSAriaIII | With 488 nm excitation (blue laser) |
| Digital Blot Scanner | LI-COR | C-DiGit | |
| Electrophoresis and Blotting Unit | Hoefer | SE300 miniVE | |
| Spectra Max 5 Plate Reader with Soft Max Pro 5.2 Software | Molecular Devices | SpectraMax 5 | |
| Patch-clamp amplifier | HEKA | EPC-10 | |
| Puller for preparing patch electrodes | Sutter Instruments | P-97 |
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