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. خلية ثقافة ملاحظة: جميع البروتوكولات الخلية زراعة يجب أن يؤديها باستخدام تقنيات زراعة الخلايا السليمة (للاشارة رؤية 3 الفصول الأولى من بيولوجيا الخلية: دليل المختبر، المجلد الأول 10). خلايا التعليم العالي-OC1 لا تتطلب أي طلاء...

<ol>
	<li>Jat, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+derivation+of+conditionally+immortall+cell+lines+from+an+H-2Kb-tsA58+transgenic+mouse.">Direct derivation of conditionally immortall cell lines from an H-2Kb-tsA58 transgenic mouse.</a> Proc. Natl. Acad. Sci. 88, 5096-5100 (1991).</li><li>Kalinec, G. M., Webster, P., Lim, D. J., Kalinec, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cochlear+cell+line+as+an+in+vitro+system+for+drug+ototoxicity+screening.">A cochlear cell line as an in vitro system for drug ototoxicity screening.</a> Audiol. Neurootol. 8, 177-189 (2003).</li><li>Devarajan, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cisplatin-induced+apoptosis+in+auditory+cells:+role+of+death+receptor+and+mitochondrial+pathways.">Cisplatin-induced apoptosis in auditory cells: role of death receptor and mitochondrial pathways.</a> Hear Res. 174, 45-54 (2002).</li><li>Chen, F. Q., Hill, K., Guan, Y. J., Schacht, J., Sha, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+apoptotic+pathways+in+the+absence+of+cell+death+in+an+inner-ear+immortomouse+cell+line.">Activation of apoptotic pathways in the absence of cell death in an inner-ear immortomouse cell line.</a> Hear Res. 284, 33-41 (2012).</li><li>Hayashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+autophagy+pathway+maintained+signaling+crosstalk+with+the+Keap1-Nrf2+system+through+p62+in+auditory+cells+under+oxidative+stress.">The autophagy pathway maintained signaling crosstalk with the Keap1-Nrf2 system through p62 in auditory cells under oxidative stress.</a> Cell Signal. 27, 382-393 (2015).</li><li>Tsuchihashi, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+through+4EBP1+and+AMPK+regulates+oxidative+stress-induced+premature+senescence+in+auditory+cells.">Autophagy through 4EBP1 and AMPK regulates oxidative stress-induced premature senescence in auditory cells.</a> Oncotarget. 6, 3644-3655 (2015).</li><li>Youn, C. K., Kim, J., Park, J. H., Do, N. Y., Cho, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+autophagy+in+cisplatin-induced+ototoxicity.">Role of autophagy in cisplatin-induced ototoxicity.</a> Int J Pediatr Otorhinolaryngol. 79, 1814-1819 (2015).</li><li>Kalinec, G., Thein, P., Park, C., Kalinec, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEI-OC1+cells+as+a+model+for+investigating+drug+cytotoxicity.">HEI-OC1 cells as a model for investigating drug cytotoxicity.</a> Hear Res. 335, 105-117 (2016).</li><li>Park, C., Thein, P., Kalinec, G., Kalinec, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEI-OC1+cells+as+a+model+for+investigating+prestin+function.">HEI-OC1 cells as a model for investigating prestin function.</a> Hear Res. 335, 9-17 (2016).</li><li>Celis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Biology: A Laboratory Handbook. 1, (2006).</li><li>Bertolaso, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+in+the+OC-k3+immortalized+cell+line+treated+with+different+agents.">Apoptosis in the OC-k3 immortalized cell line treated with different agents.</a> Audiology. 40, 327-335 (2001).</li><li>Kalinec, F., Kalinec, G., Boukhvalova, M., Kachar, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+conditionally+immortalized+organ+of+corti+cell+lines.">Establishment and characterization of conditionally immortalized organ of corti cell lines.</a> Cell Biol Int. 23, 175-184 (1999).</li><li>Belyantseva, I., Kalinec, G. M., Kalinec, F., Kachar, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+two+immortalized+cell+lines+derived+from+the+stria+vascularis+of+a+transgenic+mouse.">In vitro differentiation of two immortalized cell lines derived from the stria vascularis of a transgenic mouse.</a> 21st Midwinter Meeting Association for Research in Otolaryngology. 620a, (1998).</li><li>Gratton, M. A., Meehan, D. T., Smyth, B. J., Cosgrove, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strial+marginal+cells+play+a+role+in+basement+membrane+homeostasis:+in+vitro+and+in+vivo+evidence.">Strial marginal cells play a role in basement membrane homeostasis: in vitro and in vivo evidence.</a> Hear Res. 163, 27-36 (2002).</li><li>Debacq-Chainiaux, F., Erusalimsky, J. D., Campisi, J., Toussaint, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+to+detect+senescence-associated+beta-galactosidase+(SA-betagal)+activity,+a+biomarker+of+senescent+cells+in+culture+and+in+vivo.">Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo.</a> Nat Protoc. 4, 1798-1806 (2009).</li><li>Santos-Sacchi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+inhibition+of+voltage-dependent+outer+hair+cell+motility+and+capacitance.">Reversible inhibition of voltage-dependent outer hair cell motility and capacitance.</a> J. Neurosci. 11, 3096-3110 (1991).</li><li>Fink, S. L., Cookson, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis,+pyroptosis,+and+necrosis:+mechanistic+description+of+dead+and+dying+eukaryotic+cells.">Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells.</a> Infect Immun. 73, 1907-1916 (2005).</li><li>Majno, G., Joris, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis,+oncosis,+and+necrosis.+An+overview+of+cell+death.">Apoptosis, oncosis, and necrosis. An overview of cell death.</a> Am J Pathol. 146, 3-15 (1995).</li><li>Vanden Berghe, T., Linkermann, A., Jouan-Lanhouet, S., Walczak, H., Vandenabeele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+necrosis:+the+expanding+network+of+non-apoptotic+cell+death+pathways.">Regulated necrosis: the expanding network of non-apoptotic cell death pathways.</a> Nat Rev Mol Cell Biol. 15, 135-147 (2014).</li><li>Sun, L., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+kind+of+cell+suicide:+mechanisms+and+functions+of+programmed+necrosis.">A new kind of cell suicide: mechanisms and functions of programmed necrosis.</a> Trends Biochem Sci. 39, 587-593 (2014).</li><li>Chan, F. K., Luz, N. F., Moriwaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+necrosis+in+the+cross+talk+of+cell+death+and+inflammation.">Programmed necrosis in the cross talk of cell death and inflammation.</a> Annu Rev Immunol. 33, 79-106 (2015).</li><li>Vercammen, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+signaling+of+the+Fas+receptor:+initiation+of+both+apoptotic+and+necrotic+cell+death+pathways.">Dual signaling of the Fas receptor: initiation of both apoptotic and necrotic cell death pathways.</a> J Exp Med. 188, 919-930 (1998).</li><li>Campisi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging,+cellular+senescence,+and+cancer.">Aging, cellular senescence, and cancer.</a> Annu Rev Physiol. 75, 685-705 (2013).</li><li>Bian, S., Koo, B. W., Kelleher, S., Santos-Sacchi, J., Navaratnam, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+expressing+Tet-inducible+cell+line+recapitulates+in+situ+developmental+changes+in+prestin's+Boltzmann+characteristics+and+reveals+early+maturational+events.">A highly expressing Tet-inducible cell line recapitulates in situ developmental changes in prestin's Boltzmann characteristics and reveals early maturational events.</a> Am J Physiol Cell Physiol. 299, C828-C835 (2010).</li><li>Abe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+expression+of+the+outer+hair+cell+motor+prestin+in+the+mouse.">Developmental expression of the outer hair cell motor prestin in the mouse.</a> J Membr Biol. 215, 49-56 (2007).</li><li>Oliver, D., Fakler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+density+and+functional+characteristics+of+the+outer+hair+cell+motor+protein+are+regulated+during+postnatal+development+in+rat.">Expression density and functional characteristics of the outer hair cell motor protein are regulated during postnatal development in rat.</a> J Physiol. 519 Pt 3, 791-800 (1999).</li><li>Tsunoo, M., Perlman, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+Oxygen+Tension:+Relation+to+Blood+Flow+and+Function.">Cochlear Oxygen Tension: Relation to Blood Flow and Function.</a> Acta Otolaryngol. 59, 437-450 (1965).</li></ol>

في هذا التقرير وصفنا كيفية الثقافة خلايا التعليم العالي-OC1 واستخدامها لتقييم آليات للخلايا المخدرات التي يسببها والتحقيق في الخصائص الفنية للprestin، المحرك الجزيئي للقوقعة OHCs. الإجراءات الفنية، ومع ذلك، فإن العام بما فيه الكفاية لتتكيف بسهولة مع مختلف الدراسات. <p cl...

منزل الأذن معهد-الجهاز كورتي 1 تستمد (HEI-OC1) خلايا من الجهاز السمعي من 1،2 الماوس المعدلة وراثيا. حضانة أي خلية من هذا الفأر وراثيا في 33 ° C / 10٪ CO 2 (شروط متساهلة) يستحث تعبير عن الجينات تخليد الذي يقوم بتشغيل دي التمايز وتسارع انتشار. تحريك الخلايا إلى 39 ° C / 5٪ CO 2 (ظروف غير متساهلة) تؤدي إلى انخفاض انتشار، والتمايز، وعلى الأقل في حالة من مؤسسات التعليم العالي-OC1، موت الخلايا 2،3. تم استنساخ خلايا التعليم العالي-OC1 وتميزت في المختبر لدينا منذ أكثر من عشر سنوات، وأشارت الدراسات الأولية أنها تعبير عن علامات معينة من خلايا الشعر قوقعة، مثل prestin، الميوسين 7A، Atoh1، عامل التغذية العصبية، calbindin وكالمودولين، ولكن أيضا علامات من دعم خلايا مثل connexin 26 و نمو الخلايا الليفية مستقبلات عامل (جمعية جيل المستقبل-R) 2. لذلك، اقترح أن مؤسسات التعليم العالي-OC1 يمكن أن تمثل commoن السلف للخلايا الحسية ودعم من جهاز كورتي 2. قدمت دراسات موازية أدلة قوية على أن التوراتية المخدرات السامة مثل سيسبلاتين، جنتاميسين والستربتومايسين التي يسببها كاسباس 3 التنشيط في هذه الخلايا، في حين لم المخدرات تعتبر غير سامة للأذن، مثل البنسلين، وليس 2،3. لذلك، اقترح هذا الخط الخلية كنظام في المختبر للتحقيق في الآليات الخلوية والجزيئية تشارك في تسميم أذني وللفحص من احتمال التسمم الأذني أو خصائص otoprotective العقاقير الدوائية الجديدة. وتشير التقديرات إلى أن الخلايا مؤسسات التعليم العالي-OC1 استخدمت في أكثر من مائة وخمسين دراسة نشرت في السنوات العشر الماضية. في حين تبحث في التأثير المحتمل الموالية لأفكارك من الأدوية المختلفة كان الهدف الرئيسي لمعظم الدراسات التي تنطوي على هذا الخط الخلية، عمليات الخلية الهامة الأخرى مثل الالتهام الذاتي والشيخوخة قد بدأت لتوها إلى التحقيق في زنازين مؤسسات التعليم العالي-OC1 4-7. أنانا دراسة حديثة من مختبرنا 8، استخدمنا خلايا التعليم العالي-OC1 لجمع مجموعة شاملة من البيانات عن موت الخلايا والبقاء والانتشار، الشيخوخة والالتهام الذاتي الناجم عن العقاقير الدوائية المختلفة التي يكثر استخدامها في العيادة. نحن أيضا مقارنة بعض استجابات الخلايا مؤسسات التعليم العالي-OC1 مع تلك من كلوة-293 (خلايا الكلى الجنينية البشرية) وهيلا (خلايا الظهارية البشرية) يتلقون العلاج متطابقة. وأشارت النتائج التي توصلنا إليها خلايا التعليم العالي-OC1 تستجيب إلى كل دواء بطريقة مميزة، مع dose- مميزة والحساسية تعتمد على الوقت واحدة على الأقل من آليات قيد الدراسة. أكدنا أيضا في هذه الدراسة أن التفسير الصحيح للنتائج التجريبية سوف يتطلب إجراء دراسات موازية مع تقنية أكثر من 8. وفي دراسة مختلفة ونحن التحقيق في استخدام الخلايا مؤسسات التعليم العالي-OC1 لتقييم الاستجابة الوظيفية لprestin، البروتين السيارات من خلايا الشعر الخارجي قوقعة (OHCs) 9 </sتصل&gt;. أبلغنا التدفق الخلوي ودراسات المسح المجهري ليزر متحد البؤر على نمط من التعبير prestin، وكذلك السعة غير الخطية (NLC) والدراسات لقط خلية التصحيح كلها في الخلايا مؤسسات التعليم العالي-OC1 مثقف في متساهلة (P-HEI-OC1) وغير متساهلة (NP-HEI-OC1) الظروف. وأشارت النتائج التي توصلنا إليها كل من إجمالي التعبير prestin وغشاء البلازما زيادة توطين بطريقة تعتمد على الوقت في الخلايا NP-HEI-OC1. ومن المثير للاهتمام، وجدنا أيضا أن الزيادة في توطين prestin في الغشاء البلازمي للخلايا NP-HEI-OC1 المترابطة مع انخفاض في نا + K + أتباز، التي translocated من غشاء البلازما إلى السيتوبلازم دون تغييرات كبيرة في إجمالي التعبير الخلية. وبالإضافة إلى ذلك، أثبتنا أن الخلايا P-HEI-OC1 لها مؤتمر العمل النيجيري القوي المرتبطة prestin الوظائف الحركية، والتي انخفضت عندما زادت كثافة جزيئات prestin الحاضرة في غشاء البلازما. وإجمالا، فإن هذه النتائج تدعم بقوة فائدة مؤسسات التعليم العالي-OC1 خلايا للتحقيق البروتينات السمعية. في هذه المقالة الفيديو وصفنا كيفية الثقافة خلايا التعليم العالي-OC1، لماذا أنها مريحة لاستخدام خلايا تنمو في الظروف متساهلة (P-HEI-OC1) للدراسات السمية الخلوية، وكيفية تقييم آلية / ثانية للخلايا المخدرات التي يسببها وكيف إجراء دراسات الكهربية (على سبيل المثال، والتصحيح، المشبك، والسعة غير الخطية (NLC)) للتحقيق في الخصائص الفنية للprestin، المحرك الجزيئي للOHCs القوقعة.

<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.

في بضع من المنشورات الحديثة أبلغنا مجموعة شاملة من الدراسات التي تهدف إلى تقييم استجابة الخلايا مؤسسات التعليم العالي-OC1 لعدة عقاقير الدوائية التي يشيع استخدامها وكذلك التحقيق في وظيفة prestin 8،9. في هذه الدراسات التي قطعناها على أنفسنا استخدام ...

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.

العمل مع الخلايا السمعية مؤسسات التعليم العالي-OC1

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

وسم الخلايا الجذعية مع الأصباغ نيون للمنظمات غير الغازية مع الكشف عن التصوير الضوئي

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

تعيين آثار ما بعد من ثيتا انفجار تحفيز على القشرة السمعية الإنسان مع التصوير الوظيفي

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

الاستقراء وتحليل الظهارية الوسيطة الانتقالية ل

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

التصوير مضان مع نانومتر دقة واحدة (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

بنقل العدوى إلى خلايا RAW264.7 مع luciferase المراسل مراسل جين

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

القوارض العمل نموذج القلب لدراسة أداء عضلة القلب واستهلاك الأوكسجين

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

سم كلوي حقن مكروي في الزرد لنموذج قصور كلوي حاد

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

طرق تقدير من معدلات الجلوكوز والأحماض الدهنية الأكسدة في عزل قلب الجرذ العمل

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

واجهة المستخدم الرسومية لتتبع بمساعدة برنامج تركيز البروتين في النتوءات الخلوية الديناميكية

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

الرصد في الموقع للجمعيات النكّيّازة الجزيئية المشكلة بشكل عابر في البكتيريا والخميرة والخلايا البشرية

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 ...