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. Cellodling Obs: Alla cellodlingsprotokoll måste utföras med lämpliga cellodlingstekniker (för referens se de 3 första kapitlen i cellbiologi: A Laboratory Handbook, Volume I 10). HEI-OC1-celler kräver inte någon ytterligare beläggning eller behandling av de cellodlingsskålar för korrekt vidhäftning och tillväxt. Mycket viktigt: Använd inte glas rätter för cellodlingsändamål; fenotypen och biologiska svaret hos cellerna till farmakologiska läkemedel kommer att ä...

<ol>
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I denna rapport beskriver vi hur kulturen HEI-OC1 celler och använda dem för att utvärdera mekanismerna för läkemedelsinducerad cytotoxicitet och att undersöka funktionella egenskaper Prestin, molekylär motor cochlea OHCs. De tekniska förfaranden är dock allmänt nog att enkelt anpassas till olika studier. Alla protokoll som beskrivs här kräver korrekt användning av väletablerade cellodlingsteknik 10. Precis som med alla andra cellinje, som arbetar med HEI-OC1 celler ...

Hus Ear Institute-organ Corti en (HEI-OC1) celler är härledda från hörselorganet av en transgen mus 1,2. Inkubering av varje cell från denna transgena mus vid 33 ° C / 10% CO2 (tillåtliga betingelser) inducerar expression av en immortaliserande gen som utlöser de-differentiering och accelererad proliferation; flytta cellerna till 39 ° C / 5% CO2 (icke-tillåtande betingelser) leda till minskad proliferation, differentiering och, åtminstone i fallet med HEI-OC1, celldöd 2,3. HEI-OC1 celler klenades och karakteriserades i vårt laboratorium över ett decennium sedan, och initiala studier visade att de uttrycker specifika markörer av cochlea hårceller, såsom Prestin, myosin 7a, Atoh1, BDNF, calbindin och calmodulin, men också markörer för att stödja celler såsom connexin 26 och fibroblasttillväxtfaktorreceptor (FGF-R) 2. Därför föreslogs det att HEI-OC1 kunde representera en common stamfader för sensoriska och stödjande celler i organ Corti 2. Parallella studier visade tydligt att arketypiska ototoxiska läkemedel som cisplatin, gentamicin och streptomycin inducerade kaspas-3-aktivering i dessa celler, medan droger anses icke-ototoxiska, som penicillin, inte 2,3. Därför var denna cellinje föreslås som ett in vitro-system för att undersöka de cellulära och molekylära mekanismer som är involverade i ototoxicitet och för screening av potentiella ototoxicitet eller otoprotective egenskaper hos nya farmakologiska läkemedel. Det uppskattas att HEI-OC1 celler har använts i mer än hundra och femtio studier som publicerats under de senaste tio åren. Medan du tittar på den potentiella pro-apoptotiska effekten av olika läkemedel var viktigt mål för de flesta studier med denna cellinje har andra viktiga cellprocesser som autophagy och åldrande precis börjat utredas i HEI-OC1 celler 4-7. jagna färsk studie från vårt laboratorium 8 använde vi HEI-OC1 celler att samla en omfattande uppsättning data om celldöd, överlevnad, proliferation, åldrande och autophagy inducerad av olika farmakologiska läkemedel som ofta används vid kliniken. Vi jämförde också några av svaren från HEI-OC1-celler med de från HEK-293 (humana embryonala njurceller) och HeLa (humana epitelceller) som fick identisk behandling. Våra resultat indikerade att HEI-OC1-celler svarar på varje läkemedel på ett karakteristiskt sätt, med en distinkt dos- och tidsberoende känslighet för åtminstone en av de mekanismer som studeras. Vi betonade också i denna studie att en korrekt tolkning av de experimentella resultaten kommer att kräva att utföra parallella studier med mer än en teknik 8. I en annan studie undersökte vi användning av HEI-OC1-celler för att utvärdera den funktionella responsen hos Prestin, motor proteinet av cochlea yttre hårceller (OHCs) 9 </supp&gt;. Vi rapporterade flödescytometri och konfokal laserscanningsmikroskopi studier på mönstret av Prestin uttryck, liksom icke-linjär kapacitans (NLC) och hela cell patch klämstudier i HEI-OC1 celler odlade vid tillåtande (P-HEI-OC1) och icke- tillåtande (NP-HEI-OC1) förhållanden. Våra resultat indikerade att både total Prestin uttryck och plasmamembranlokalisering ökning av ett tidsberoende sätt i NP-HEI-OC1-celler. Intressant nog fann vi också att ökningen i Prestin lokalisering vid plasmamembranet av NP-HEI-OC1-celler korrelerade med en minskning av Na + K + ATPas, som translokeras från plasmamembranet till cytoplasman utan betydande förändringar i den totala celluttryck. Dessutom visade vi att P-HEI-OC1-celler har en robust NLC associerade till Prestin motorik, som minskade när densiteten hos Prestin molekyler som är närvarande vid plasmamembranet ökas. Sammantaget stöder dessa resultat starkt nyttan av HEI-OC1-celler för att undersöka hörsel proteiner. I denna video artikeln beskriver vi hur kulturen HEI-OC1 celler, varför det är lämpligt att använda celler växer tillåtande betingelser (P-HEI-OC1) för cytotoxicitet studier, hur man ska värdera mekanismen / s av läkemedelsinducerad cytotoxicitet och hur för att utföra elektrofysiologiska studier (t.ex., patch-clamp, icke-linjär kapacitans (NLC)) för att undersöka funktionella egenskaperna hos Prestin, den molekylära motorn av cochlea OHCs.

<table border="0" cellpadding="0" cellspacing="0" width="1447">
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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.

I ett par av de senaste publikationer rapporterade vi en omfattande uppsättning studier som syftar till att utvärdera svaret från HEI-OC1 celler till flera vanliga farmakologiska narkotika samt undersöka Prestin funktion 8,9. I dessa studier gjorde vi användning av alla de protokoll som beskrivs i tidigare avsnitt. Ett av resultaten av dessa tidigare studier var att HEI-OC1-celler odlades vid icke tillåtliga b...

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.

Arbeta med Auditory HEI-OC1 celler

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

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Märkning stamceller med fluorescerande färger för icke-invasiv Detection med optisk avbildning

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

Kartläggning av efterverkningarna av Theta skurstimuleringen om den mänskliga hörselbarken med funktionella Imaging

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

Induktion och Analys av Epithelial till mesenkymala övergång

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

Fluorescens Imaging med One-nanometer Noggrannhet (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

Transfektera RAW264.7 Celler med en luciferasrapportörgen

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

Gnagare Working Heart modell för studier av Myocardial prestanda och syreförbrukning

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

Nefrotoxin mikroinjektion i Zebrafish till Model akut njursvikt

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

Metoder för bestämning av priser av glukos och fettsyra oxidation i isolerade Working Rat hjärta

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

En grafisk användargränssnitt för programvaruassisterad spårning av proteinkoncentration i dynamiska cellulära protrusioner

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

In situ övervakning av transitivt bildade molekylär Chaperone församlingar i bakterier, jäst, och mänskliga celler

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 ...