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. Zellkultur Hinweis: Alle Zellkulturprotokolle müssen unter Verwendung geeigneter Zellkulturtechniken durchgeführt werden (als Referenz siehe die ersten drei Kapitel der Zellbiologie: A Laboratory Handbook, Band I 10). HEI-OC1-Zellen benötigen keine zusätzliche Beschichtung oder Behandlung der Zellkulturschalen für eine ordnungsgemäße Anhaftung und Wachstum. Sehr wichtig: Verwenden Sie keine Glaswaren Geschirr für die Zellkultur Zwecke; der Phänotyp und biologische Reakti...

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In diesem Bericht beschreiben wir, wie sie zur Kultur HEI-OC1-Zellen und verwenden Mechanismen der medikamenteninduzierten Zytotoxizität zu bewerten und funktionellen Eigenschaften von Prestin, der molekulare Motor der Cochlea-OHC zu untersuchen. Die technischen Verfahren sind jedoch allgemein genug, um leicht an verschiedene Studien anzupassen. Alle Protokolle hier beschrieben sind, erfordern die korrekte Verwendung von gut etablierten Zellkulturtechniken 10. Genau wie bei jedem...

House Ear Institute-Corti - Organ 1 (HEI-OC1) Zellen werden aus dem Hörorgan einer transgenen Maus 1,2 abgeleitet. Inkubation einer beliebigen Zelle aus dieser transgenen Maus , bei 33 ° C / 10% CO 2 (permissive Bedingungen) induziert die Expression eines Gens , das Immortalisieren Entdifferenzierung und beschleunigter Proliferation auslöst; Bewegen der Zellen auf 39 ° C / 5% CO 2 (nicht-permissive Bedingungen) führen zu einer verminderten Proliferation, Differenzierung und zumindest im Fall von HEI-OC1, Zelltod 2,3. HEI-OC1-Zellen wurden in unserem Labor vor über einem Jahrzehnt kloniert und charakterisiert, und die anfängliche Studien gezeigt, dass sie spezifische Marker der Cochlea-Haarzellen, wie prestin, Myosin 7a, Atoh1, BDNF, Calbindin und Calmodulin exprimieren, sondern auch Marker der Stütz Zellen wie Connexin 26 und Fibroblasten - Wachstumsfaktor - Rezeptor (FGF-R) 2. Daher wurde vorgeschlagen, dass HEI-OC1 eine commo darstellen könnten Vorläufer für sensorische und Stützzellen des Corti - Organ 2. Parallel Studien lieferten starke Hinweise darauf , dass ototoxic Medikamente wie Cisplatin, Gentamicin und Streptomycin induzierte Caspase-3 - Aktivierung in diesen Zellen archetypischen, während Medikamente als nicht-ototoxische, wie Penicillin, nicht 2,3. Daher wurde diese Zelllinie als ein in vitro - System vorgeschlagen , um die zellulären und molekularen Mechanismen in Ototoxizität beteiligt zu untersuchen , und für das Screening des potentiellen Ototoxizität oder otoprotektiven Eigenschaften neuer pharmakologischer Wirkstoffe. Es wird geschätzt, dass HEI-OC1-Zellen wurden in mehr als einhundertfünfzig Untersuchungen in den letzten zehn Jahren veröffentlicht verwendet. Während der Suche das Hauptziel der meisten Studien war auf dem Potential pro-apoptotische Wirkung verschiedener Medikamente beteiligt diese Zelllinie, andere wichtige Zellprozesse wie Autophagie und Seneszenz haben gerade erst begonnen 4-7 in HEI-OC1 - Zellen untersucht werden. ichna aktuelle Studie aus unserem Labor 8 verwendeten wir HEI-OC1 - Zellen einen umfassenden Satz von Daten über den Zelltod, Überleben, Proliferation, Seneszenz und durch unterschiedliche pharmakologische Wirkstoffe in der Klinik verwendet häufig induzierten Autophagie zu sammeln. Wir verglichen auch einige der Antworten von HEI-OC1-Zellen mit denen aus HEK-293 (menschliche embryonale Nierenzellen) und HeLa (humane Epithelzellen) Empfangen identische Behandlung. Unsere Ergebnisse zeigten, dass HEI-OC1 Zellen an die jedes Medikament in einer charakteristischen Art und Weise, mit einem unverwechselbaren dosis- und zeitabhängige Empfindlichkeit auf mindestens einer der Mechanismen zu untersuch reagieren. Wir betonten auch in dieser Studie , dass eine korrekte Interpretation der experimentellen Ergebnisse erfordern parallel Studien mit mehr als eine Technik 8 durchgeführt wird . In einer anderen Studie untersuchten wir die Verwendung von HEI-OC1 - Zellen die funktionelle Reaktion von Prestin, das Motorprotein von Cochlea äußeren Haarzellen (OHC) 9 zu bewerten </sup&gt;. Wir berichteten Durchflusszytometrie und der konfokalen Laser-Scanning-Mikroskopie Studien über die Muster von Prestin Ausdruck, sowie nichtlineare Kapazität (NLC) und Ganzzell-Patch-Clamp-Untersuchungen in HEI-OC1-Zellen, die in permissiven (P-HEI-OC1) und nicht permissive (NP-HEI-OC1) Bedingungen. Unsere Ergebnisse zeigten, dass sowohl die Gesamt prestin Expression und Plasmamembran-Lokalisations Anstieg in einer zeitabhängigen Weise in NP-HEI-OC1-Zellen. Interessanterweise fanden wir auch , dass der Anstieg in prestin Lokalisierung in der Plasmamembran von NP-HEI-OC1 - Zellen mit einer Abnahme korreliert in Na + K + ATPase, welche von der Plasmamembran zum Zytoplasma ohne wesentliche Änderungen in Gesamtzellexpressions transloziert. Darüber hinaus haben wir gezeigt, dass P-HEI-OC1-Zellen haben eine robuste NLC Motorfunktion prestin verbunden, die abnimmt, wenn die Dichte der prestin Moleküle, die an der Plasmamembran erhöht. Insgesamt stark diese Ergebnisse die Nützlichkeit von HEI-OC unterstützen1-Zellen auditorischen Proteine ​​zu untersuchen. In diesem Video-Artikel beschreiben wir, wie HEI-OC1-Zellen zu Kultur, warum es bequem ist, Zellen zu verwenden, die an der permissiven Bedingungen (P-HEI-OC1) für Zytotoxizitätsuntersuchungen, wie der Mechanismus / s von arzneimittelinduzierten Zytotoxizität zu bewerten und wie elektro~~POS=TRUNC Untersuchungen (zB Patch-clamp, nicht-lineare Kapazität (NLC)) durchzuführen funktionellen Eigenschaften von prestin, der molekularen Motor Cochlea OHCs zu untersuchen.

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	<tbody>
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			<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.

In ein paar jüngsten Veröffentlichungen berichteten wir eine umfassende Reihe von Studien zur Bewertung der Reaktion von dem Ziel HEI-OC1 - Zellen auf mehrere häufig verwendete pharmakologische Wirkstoffe sowie die Untersuchung prestin Funktion 8,9. In diesen Studien haben wir die Verwendung aller Protokolle in den vorhergehenden Abschnitten beschrieben. Eines der Ergebnisse dieser früheren Studien war , dass HE...

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.

Arbeiten mit Auditory HEI-OC1-Zellen

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.

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