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. Cell Culture
Note: All cell culturing protocols must be performed using proper cell culture techniques (for reference see the first 3 Chapters of Cell Biology: A Laboratory Handbook, Volume I 10). HEI-OC1 cells do not require any additional coating or treatment of the cell culture dishes for proper adherence and growth. Very important: do not use glassware dishes for cell culture purposes; the phenotype and biological response of the cells to pharmacological drugs will change (G Kalinec &#38; F Kalinec, unpublished); conventional plastic cell culture dishes are recommended (see Table of Materials/Equipment). Pay special attention to aseptic techniques to avoid contaminations, but never use antibiotics (e.g., ampicillin or streptomycin) with HEI-OC1 cells. If necessary, use amphotericin B. While HeLa and HEK-293 cells have been used as control in previous studies with HEI-OC1 8, any other cell line could be acceptable for this purpose.
<ol>
	<li>Resuscitation of Frozen HEI-OC1 Cells 
	Note: This protocol can also be used with other cell lines from the same transgenic mouse developed in our laboratory, such as OC-k3, from organ of Corti 11,12, and SV-k1, from stria vascularis 13,14.
	<ol>
		<li>Remove a vial of cryopreserved HEI-OC1 cells from liquid N2, and place it in a water bath at 37 &#176;C. Submerge only the lower half of the vial, and allow it to thaw until only a small amount of ice remains in the vial. Wipe the outside of the vial with 70% alcohol.</li>
		<li>Pipette the cells from the vial and deliver the entire volume (~ 1.5 x 105 cells/ml) slowly, drop by drop, into a 15 ml conical tube containing 10 ml of a pre-warmed growth medium, like Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), supplemented with 10% fetal bovine serum (FBS).</li>
		<li>Remove DMSO by centrifuging the tube at 300-500 x g for 5 min, discarding the supernatant and re-suspending the cells in 9 ml of fresh growth medium (DMEM + 10% FBS). Disaggregate clumps or sheets of cells by flux and reflux of the suspended cells, from the pipette to the medium and vice-versa, three to four times with the tip of the pipette touching the bottom of the tube.</li>
		<li>Place the total volume of cells suspended in growth medium in 100 mm-diameter untreated, plastic cell culture dishes.</li>
		<li>Incubate the cells at 33 &#176;C with 10% CO2 (permissive conditions).</li>
	</ol>
	</li>
	<li>Subculture of HEI-OC1 Cells 
	Note: Prior to confluence (~80%) the cells should be brought into suspension and sub-cultured in order to prevent the culture dying. This protocol can also be used with other cell lines developed in our laboratory from the same transgenic mouse, such as OC-k3, from organ of Corti 11,12, and SV-k1, from stria vascularis 13,14.
	<ol>
		<li>Remove old medium and wash the cells with 2 ml of PBS.</li>
		<li>Cover the cell monolayer with a solution of 0.25% trypsin, using 1 ml per 25 cm2 of surface area. To move onto the next step more than 40% of the cells must be detached. Examine the cells using an inverted microscope and, if necessary, &#34;slap or tap&#34; the culture dishes gently to release any remaining attached cells. 
		Note: Take care as trypsinization for long periods can cause cell death; not enough and the transferred cells will insufficient for the planned studies.</li>
		<li>Resuspend the cells, following the procedure described in 1.1.3, and transfer them to a 15 ml conical tube.</li>
		<li>Centrifuge for 5 min at 300-500 x g, discard the medium, collect the cells with fresh growth medium and seed them in, at least, four 100 mm-diameter cell culture dishes. The number of cells per dish will depend on the amount of cells recovered. Incubate the cells at 33 &#176;C with 10% CO2 (P-HEI-OC1) and divide again as many times as necessary for the planned experiments or for generating a new stock.</li>
		<li>For stock preparation, replace 100 mm-diameter dishes by 250-550 ml cell culture flasks; for experiments, smaller dishes or multi-wells plates may be more adequate.</li>
		<li>For differentiation, incubate HEI-OC1 cells at permissive conditions until they reach ~80% confluence; then move the dishes to 39 &#176;C with 5% CO2 (NP-HEI-OC1) for 2 to 3 weeks. 
		Note: Cells will progressively stop proliferating and dying. Change the medium every other day to remove dead cells. Depending on the original number of cells, usually after ~ 4 weeks no more cells will be available for experiments. Differentiation starts as soon as the cells are placed at NP conditions, but not every cell differentiate at the same pace. Importantly, prestin expression and membrane localization increases during the differentiation process (see Representative Results, Figure 4), providing a qualitative and quantitative indication of the level of differentiation.</li>
	</ol>
	</li>
</ol>
2. Drug Cytotoxicity Studies
Note: HEI-OC1 cells grown at permissive conditions (P-HEI-OC1) are recommended for these studies (see Representative Results, Figure 1).
<ol>
	<li>Cell Viability (MTT Assay)
	<ol>
		<li>Collect P-HEI-OC1 cells by following the procedure described in 1.2, and count them with either an automatic cell counter or hemocytometer. Adjust the concentration to 2.0 x 105 cells/ml.</li>
		<li>Seed the cells on 96-well clear flat bottom plates (100 &#181;l per well), incubate overnight for attachment to the substrate, and then treat them with the drugs of interest. Do not forget to include blanks and non-treated cells (control)!</li>
		<li>After drug treatment (usually 24 or 48 hr at 33 &#176;C), perform the MTT assay following the manufacturer&#39;s protocol. In general, the protocols for this assay have the following steps:
		<ol>
			<li>Add 10 &#181;l of MTT reagent to each well.</li>
			<li>Incubate the plate at 37 &#176;C for 2 to 4 hr until purple dye is visible, and then add 100 &#181;l of the MTT Detergent Reagent to each well. Do not shake the plate.</li>
			<li>Cover the plate and leave it in the dark for 2 to 4 hr at 37 &#176;C.</li>
			<li>Use a microplate plate reader to measure absorbance at 570 nm in each well, including the blanks (growth medium alone). Normalize data using the average OD in control cells as 100% of viability.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Caspase 3/7 Activation Assay 
	Note: HEI-OC1 cells grown at permissive conditions (P-HEI-OC1) are recommended for these studies.
	<ol>
		<li>Collect HEI-OC1 cells by following the procedure described in 1.2, and count them with either an automatic cell counter or a hemocytometer. Adjust the concentration to 2.0 x 105 cells/ml.</li>
		<li>Seed the cells on white-walled 96-well plates (100 &#181;l per well), incubate overnight for attachment to the substrate, and then treat them with the drugs of interest. Do not forget to include blanks and non-treated cells (control)!</li>
		<li>After drug treatment (usually 24 or 48 hr at 33 &#176;C), perform the caspase assay following the respective manufacturer&#39;s protocol. In general, the protocols for this assay have the following steps:
		<ol>
			<li>Prepare the reagents, mix them well, and allow them to equilibrate to room temperature.</li>
			<li>Remove the cells from the incubator and allow the plates to equilibrate to room temperature.</li>
			<li>Add the indicated amount of reagent to each well, being careful to avoid cross-contamination by not touching wells containing different samples with the same pipette tips.</li>
			<li>Cover the plate and mix for 30 sec using a plate shaker at 300-500 rpm.</li>
			<li>Incubate the plate at room temperature for a period of, at least, 30 min. Determine the optimal incubation period empirically. If the room temperature fluctuates use a constant-temperature incubator, because temperature fluctuations will affect luminescence reading.</li>
			<li>Measure luminescence in each well, and normalize values using average luminescence in control cells as 100% of caspase activation.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cell Division (Proliferation)
	<ol>
		<li>Collect HEI-OC1 cells by following the procedure described in 1.2, and count them with either an automatic cell counter or a hemocytometer. Adjust the concentration to 2.0 x 105 cells/ml.</li>
		<li>Seed the cells on 96-well clear flat bottom plates (100 &#181;l per well), incubate overnight at permissive conditions for attachment to the substrate, and then treat them with the drugs of interest. Don&#39;t forget to include blanks and non-treated cells (control)!</li>
		<li>One hour after treatment, add to each well 1 &#181;l of prepared 100x BrdU (5-Bromo-2&#8242;-deoxyuridine) solution, and return the plates to the incubator at 33 &#176;C.</li>
		<li>After 12, 24 and 48 hr, remove the existing medium from the corresponding plates and wash each well once with PBS.</li>
		<li>Perform the cell proliferation assay following the manufacturer&#39;s protocol. In general, the protocols for this assay have the following steps:
		<ol>
			<li>Prepare the reagents, including fixing/denaturing solution, wash buffer, primary antibody detection solution, secondary antibody detection solution, and BrdU solution as indicated in the manufacturer&#39;s protocol.</li>
			<li>Add the fixing/denaturing solution to each well, in the amounts indicated in the manufacturer&#39;s protocol, and keep the plate at room temperature for 30 min.</li>
			<li>Remove the fixing/denaturing solution and add the primary antibody at the concentration indicated in the manufacturer&#39;s protocol. Keep the plate at room temperature for 1 hr.</li>
			<li>Remove the solution with the primary antibody, wash the plate 3 times with wash buffer, and then add the secondary antibody at the concentration indicated in the manufacturer&#39;s protocol. Keep the plate with the secondary antibody at room temperature for 30 min.</li>
			<li>Remove the solution with the secondary antibody, wash the plate 3 times with wash buffer, and add the substrate. After 10 min incubation at room temperature, add the STOP solution. Be careful: Control the change in color; if the solution becomes very dark, stop the reaction prior to the standard development time of 10 min.</li>
			<li>Read absorbance at 450 nm within 30 min of adding the STOP Solution.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cytotoxicity 
	Note: HEI-OC1 cells grown at permissive conditions (P-HEI-OC1) are recommended for these studies.
	<ol>
		<li>Incubate at permissive conditions, usually for 24-48 hr, HEI-OC1 cells in cell culture medium alone (control) or medium plus the drugs of interest.</li>
		<li>At the end of the treatment, wash the cells three times with PBS, and detach using 1 ml per 25 cm2 of surface area of a non-enzymatic cell dissociation solution for 3 min.</li>
		<li>After detaching, collect and pellet the cells by centrifugation at 3,000 x g for 5 min, remove the supernatant, and stain the cells for 15 min in the dark with the reagent included in cytotoxicity assay.</li>
		<li>Determine the number of live HEI-OC1 cells by flow cytometry as indicated by the manufacturer of the flow cytometer.</li>
	</ol>
	</li>
	<li>Senescence
	<ol>
		<li>Incubate at permissive conditions, usually for 24-48 hr, HEI-OC1 cells in cell culture medium alone (control) or medium plus the drugs of interest.</li>
		<li>Determine the number of senescence-associated beta-galactosidase positive cells using flow cytometry with the protocol described by Debacq-Chainiaux et al. 15 or other method known to provide reliable results. A brief protocol for flow cytometry is the following:
		<ol>
			<li>At the end of the experimental treatments, wash the cells three times with PBS and then incubate them with 100 nM Bafilomycin A1 in cell culture medium for 1 hr at permissive conditions.</li>
			<li>Add C12FDG at a final concentration of ~33 &#181;M, and continue incubating the cells for 1-2 hr.</li>
			<li>Wash the cells twice with PBS, harvest them using 1 ml per 25 cm2 of surface area of a non-enzymatic cell dissociation solution for 3 min, and pellet them by centrifugation at 3,000 x g for 5 min.</li>
			<li>Resuspend the cells in ice-cold PBS and determine the number of positive HEI-OC1 cells by flow cytometry as indicated by the manufacturer of the flow cytometer.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Autophagy
	<ol>
		<li>Collect HEI-OC1 cells control and starved (deprived of serum for 48 hr) by following the procedure described in 1.2, and process them for Western blot following standard protocols. In general, the protocols for Western blot have the following steps:
		<ol>
			<li>Seed HEI-OC1 cells in 6-well plates at a concentration of 1.0 x 105 cells/ml. After 24 and/or 48 hr, wash the cells with ice-cold PBS.</li>
			<li>Lyse with 200 &#181;l of TNESV buffer (1% NP40, 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 2mM EDTA, 1 mM Na3VO4) with protease inhibitor cocktail and 1 mM PMSF for 5 min on ice.</li>
			<li>Collect the cells (by scraping the bottom of each well), transfer to microcentrifuge tubes and centrifuge at 16,800 x g for 5 min at 4 &#176;C.</li>
			<li>Collect the supernatants and quantify the protein concentration using the BCA Assay.</li>
			<li>Add 5x loading buffer and 1 mM DTT to each sample and boil for 5 min to denature the proteins.</li>
			<li>Run the samples using SDS-PAGE on a 4-12% gel, and transfer to PVDF membranes.</li>
			<li>Block the membranes with 5% nonfat dried milk in TBS-T with 0.1% Tween 20 for 60 min at RT.</li>
			<li>Incubate the membranes overnight at 4 &#176;C with primary antibodies.</li>
			<li>The next day, wash the membranes extensively with TBS-T, and incubate with a secondary IgG antibody conjugated to horseradish peroxidase (HRP) (1:1,500) for 1 hr.</li>
			<li>Detect immunoreactivity with an enhanced chemiluminescence (ECL) detection system. Normalize protein expression levels using GAPDH (1:2,000 in TBS-T with in 10% nonfat dried milk) as standard.</li>
		</ol>
		</li>
		<li>In the Western blots investigate the expression of, at least, two different markers of autophagy such as Beclin-1 and LC3B (usually at 1:1,000 dilution in TBS-T with 2.5% BSA). Do not forget to look for a control of protein expression such as GAPDH or actin.</li>
		<li>Evaluate the expression of the protein of interest by densitometry using a digital Blot scanner or computer software such as the public domain NIH Image or ImageJ (http://rsb.info.nih.gov/nih-image/).</li>
	</ol>
	</li>
</ol>
3. Electrophysiology Experiments with HEI-OC1 Cells
<ol>
	<li>Harvesting Cells 
	Note: Use cells growing in 100 mm diameter culture dishes at 50%-80% confluency. Trypsin, Accutase, enzyme-free solution, or phosphate- buffered saline + ethylene diamine tetra acetic acid (PBS-EDTA) may be used for lifting the cells without significant effects on the number and quality of the gigaseals. In some cases, however, trypsin treatment makes the cells more fragile. In these cases, allow the cells to recover for approximately 30 minutes before starting the experiments.
	<ol>
		<li>Wash the cells twice with 10 ml PBS without Ca2+ and Mg2+.</li>
		<li>Add 2 ml of an enzyme-free detacher solution, such as non-enzymatic cell dissociation solution, and incubate the dishes for 3 min at 37 &#176;C with 5% CO2.</li>
		<li>Use an inverted microscope to check the detachment of the cells. If necessary, move the cell culture dish to detach more cells, but do it gently. 
		Note: Do not shake the dish.</li>
		<li>Add 10 ml of DMEM+10% FBS and pipette the cells gently up and down five times with a 10-ml pipette.</li>
		<li>Look at the cells under a microscope. If &#62; 80% of the cells are already separated (single), no further pipetting is needed. If the cells are still in clusters, repeat the gently pipetting until more than 80% cells are single.</li>
		<li>Place the ~10 ml of suspended cells into a 15 ml conical tube, centrifuge for 2 min at 100 x g, and discard the supernatant.</li>
		<li>Use ~200 &#181;l of external recording solution (Leibovitz&#39;s L-15 medium adjusted to 305 - 310 mOsm with distilled water) to resuspend the cells. An optical control of the cells should show many single, round cells with smooth membrane edges.</li>
	</ol>
	</li>
	<li>Patch-clamp and NLC Measurements
	<ol>
		<li>Perform patch-clamp and measurements of voltage-dependent nonlinear capacitance (NLC) using adequate amplifiers and software, as well as standard electrophysiological techniques. As internal (intrapipette) solution use 150 mM KCl, 1 mM MgCl2, 0.1 mM EGTA, 2 mM ATP-Mg, 0.1 mM GTP-Na, and 10 mM HEPES; adjust pH to 7.2 with Tris.</li>
		<li>Under the microscope, select a single, healthy cell, with round and smooth membrane edges. Attach the tip of the glass pipette to the cell surface and check membrane resistance. This should be around 3-6 megaOhms, corresponding to an internal diameter (ID) of the tip of the pipette of ~2 &#181;m.</li>
		<li>Gently press the micropipette tip against the cell plasma membrane and then apply suction. A portion of the cell membrane will be suctioned into the pipette, generating an electrical resistance in the order of 10 - 100 gigaOhms (gigaseal).</li>
		<li>Establish the whole-cell condition by disrupting the plasma membrane with suction pulses.</li>
		<li>Use cells that do not express prestin, such as HEK-293, as a control 9. 
		Note: Current responses should be filtered at 5 kHz, and corrections made for the effects of residual series resistance. All data collection and most analyses can be performed using the software usually included with the lock-in amplifiers. Capacitance function can be fitted to the first derivative of a two state Boltzmann function relating nonlinear charge to membrane voltage 16. 
		 
		&#160;</li>
	</ol>
	</li>
</ol>

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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>

The authors declare no existing or potential conflict of interest.

In this report we describe how to culture HEI-OC1 cells and use them to evaluate mechanisms of drug-induced cytotoxicity and to investigate functional properties of prestin, the molecular motor of cochlear OHCs. The technical procedures, however, are general enough to be easily adapted to different studies.
All the protocols described here require the correct use of well-established cell culture techniques 10. Just like with any other cell line, working with HEI-OC1 cells requires a...

House Ear Institute-Organ of Corti 1 (HEI-OC1) cells are derived from the auditory organ of a transgenic mouse 1,2. Incubation of any cell from this transgenic mouse at 33 &#176;C/10% CO2 (permissive conditions) induces expression of an immortalizing gene that triggers de-differentiation and accelerated proliferation; moving the cells to 39 &#176;C/5% CO2 (non-permissive conditions) lead to decreased proliferation, differentiation and, at least in the case of HEI-OC1, cell death 2,3.
HEI-OC1 cells were cloned and characterized in our laboratory over a decade ago, and initial studies indicated that they express specific markers of cochlear hair cells, such as prestin, myosin 7a, Atoh1, BDNF, calbindin and calmodulin, but also markers of supporting cells like connexin 26 and fibroblast growth factor receptor (FGF-R) 2. Therefore, it was suggested that HEI-OC1 could represent a common progenitor for sensory and supporting cells of the organ of Corti 2. Parallel studies provided strong evidence that archetypal ototoxic drugs like cisplatin, gentamicin and streptomycin induced caspase-3 activation in these cells, while drugs considered non-ototoxic, like penicillin, did not 2,3. Therefore, this cell line was proposed as an in vitro system to investigate the cellular and molecular mechanisms involved in ototoxicity and for screening of the potential ototoxicity or otoprotective properties of new pharmacological drugs. It is estimated that HEI-OC1 cells have been used in more than one hundred and fifty studies published in the last ten years.
Whereas looking at the potential pro-apoptotic effect of different drugs was the major goal of most of the studies involving this cell line, other important cell processes like autophagy and senescence have just started to be investigated in HEI-OC1 cells4-7. In a recent study from our laboratory 8, we used HEI-OC1 cells to collect a comprehensive set of data about cell death, survival, proliferation, senescence and autophagy induced by different pharmacological drugs frequently used in the clinic. We also compared some of the responses of HEI-OC1 cells with those from HEK-293 (human embryonic kidney cells) and HeLa (human epithelial cells) receiving identical treatment. Our results indicated that HEI-OC1 cells respond to the each drug in a characteristic way, with a distinctive dose- and time-dependent sensitivity to at least one of the mechanisms under study. We also emphasized in that study that a correct interpretation of the experimental results will require performing parallel studies with more than one technique 8.
In a different study we investigated the use of HEI-OC1 cells to evaluate the functional response of prestin, the motor protein of cochlear outer hair cells (OHCs) 9. We reported flow cytometry and confocal laser scanning microscopy studies on the pattern of prestin expression, as well as nonlinear capacitance (NLC) and whole cell-patch clamping studies in HEI-OC1 cells cultured at permissive (P-HEI-OC1) and non-permissive (NP-HEI-OC1) conditions. Our results indicated that both total prestin expression and plasma membrane localization increase in a time-dependent manner in NP-HEI-OC1 cells. Interestingly, we also found that the increase in prestin localization at the plasma membrane of NP-HEI-OC1 cells correlated with a decrease in Na+K+ATPase, which translocated from the plasma membrane to the cytoplasm without significant changes in total cell expression. In addition, we demonstrated that P-HEI-OC1 cells have a robust NLC associated to prestin motor function, which decreased when the density of prestin molecules present at the plasma membrane increased. Altogether, these results strongly support the usefulness of HEI-OC1 cells to investigate auditory proteins.
In this video article we describe how to culture HEI-OC1 cells, why it is convenient to use cells growing at permissive conditions (P-HEI-OC1) for cytotoxicity studies, how to evaluate the mechanism/s of drug-induced cytotoxicity and how to perform electrophysiological studies (e.g., patch-clamp, non-linear capacitance (NLC)) to investigate functional properties of prestin, the molecular motor of cochlear OHCs.

<table><tbody><tr><td>HEI-OC1 cells</td><td></td><td></td><td>ALL THE ASSAY KITS, EQUIPMENTS&amp;nbsp;</td></tr><tr><td>Class II Biological Safety cabinet</td><td>The Baker Company</td><td>Sterilgard III</td><td>AND COMPANIES INDICATED IN THE</td></tr><tr><td>Refrigerated centrifuge</td><td>Eppendorf</td><td>5810R</td><td>PREVIOUS 2 COLUMNS ARE ONLY</td></tr><tr><td>Inverted microscope</td><td>Zeiss</td><td>Axiovert 25</td><td>EXAMPLES, AND ANY OTHER SIMILAR</td></tr><tr><td>Waterbath</td><td>Stovall</td><td>HWB115</td><td>PRODUCT COULD BE USED.</td></tr><tr><td>Cell counter</td><td>Nexcelom</td><td>Cellometer Auto T4</td><td></td></tr><tr><td>Two (2) Cell incubators, one at 33 &amp;deg;C/10% CO&lt;sub&gt;2&lt;/sub&gt; and other at 39 &amp;deg;C/5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Forma Scientific</td><td>3110</td><td></td></tr><tr><td>Cell culture dishes, PS, 100 mm x 20 mm with vents</td><td>Greinier Bio-One</td><td>664-160</td><td></td></tr><tr><td>Cell culture dishes , PS,&amp;nbsp; 60 mm x 15 mm with vents&amp;nbsp;</td><td>Greiner Bio-One</td><td>628160</td><td></td></tr><tr><td>Cellstar tissue culture flasks&amp;nbsp; 250 ml</td><td>Greiner Bio-One</td><td>658-175</td><td></td></tr><tr><td>Cellstar tissue cultur&amp;nbsp; flasks 550 ml</td><td>Greiner Bio-One</td><td>660-175</td><td></td></tr><tr><td>&amp;nbsp;6 well cell culture plate, with lid-Cellstar</td><td>Greiner Bio-One</td><td>657-160</td><td></td></tr><tr><td>Microtest Tissue culture plate, 96 well, flat bottom with lid</td><td>Becton Dickinson</td><td>353072</td><td></td></tr><tr><td>Micro-Assay-Plate, Chimmey, 96-well white, clear bottom</td><td>Greiner Bio-One</td><td>655098</td><td></td></tr><tr><td>50 ml Polypropylene conical tube with cap Cellstars</td><td>Becton Dickinson&amp;nbsp;</td><td>352070</td><td></td></tr><tr><td>15 ml Polypropylene conical tubes with cap-Cellstars</td><td>Greiner Bio-One</td><td>188-271</td><td></td></tr><tr><td>PBS pH 7.4 (1x)&amp;nbsp;</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td>Life Technologies</td><td>11965-084</td><td></td></tr><tr><td>Fetal bovine serum (FBS)&amp;nbsp;</td><td>Hyclone</td><td>SH10073.1</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 Medium, no phenol red</td><td>Gibco/Invitrogen</td><td>21083-027</td><td></td></tr><tr><td>Trypsin, 0.25%&amp;nbsp;</td><td>Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>TACS MTT Cell Proliferation Assay Kit</td><td>Trevigen</td><td>4890-25-K</td><td></td></tr><tr><td>Caspase-Glo 3/7 Assay&amp;nbsp; kit</td><td>Promega</td><td>&amp;nbsp; G8091&amp;nbsp;</td><td></td></tr><tr><td>BrdU Cell Proliferation Assay Kit&amp;nbsp;</td><td>Cell Signaling</td><td>6813</td><td></td></tr><tr><td>Non-enzymatic cell dissociation solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C5789</td><td></td></tr><tr><td>Cell-Tox Green Cytotoxicity Assay Kit</td><td>Promega</td><td>&amp;nbsp; G8741&amp;nbsp;</td><td></td></tr><tr><td>FACSAriaIII instrument&amp;nbsp;</td><td>BD Biosciences</td><td>&amp;nbsp;FACSAriaIII</td><td>With 488 nm excitation (blue laser)</td></tr><tr><td>Digital Blot Scanner</td><td>LI-COR</td><td>C-DiGit</td><td></td></tr><tr><td>Electrophoresis and Blotting Unit</td><td>Hoefer</td><td>SE300 miniVE</td><td></td></tr><tr><td>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><td>Patch-clamp amplifier</td><td>HEKA</td><td>EPC-10</td><td></td></tr><tr><td>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 a couple of recent publications we reported a comprehensive set of studies aimed at evaluating the response of HEI-OC1 cells to several commonly used pharmacological drugs as well as investigating prestin function 8,9. In these studies we made use of all the protocols described in the previous sections.
One of the results of these previous studies was that HEI-OC1 cells cultured at non-permissive conditions (39 &#1...

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.

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 ...

Research

JoVE Journal

Biology

14.9K Views.  University of California, Los Angeles. 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.

Video: Working with Auditory HEI-OC1 Cells

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse 1-4. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential 5. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes 1, 4, 6-11. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood.
The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 &mu;m in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly 2. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.

Whole-cell patch-clamp recordings from auditory nerve fiber dendrites at the inner hair cell ribbon synapse in the mammalian cochlea.

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the ...

Neuroscience

Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives from their characteristic apical bundle of stereocilia, a critical element for detection and transduction of sound energy 1. OHCs are able to change shape &#x2014;elongate, shorten and bend&#x2014; in response to electrical, mechanical and chemical stimulation, a motor response considered crucial for cochlear amplification of acoustic signals 2.
OHC stimulation induces two different motile responses: i) electromotility, a.k.a fast motility, changes in length in the microsecond range derived from electrically-driven conformational changes in motor proteins densely packed in OHC plasma membrane, and ii) slow motility, shape changes in the millisecond to seconds range involving cytoskeletal reorganization 2, 3. OHC bending is associated with electromotility, and result either from an asymmetric distribution of motor proteins in the lateral plasma membrane, or asymmetric electrical stimulation of those motor proteins (e.g., with an electrical field perpendicular to the long axis of the cells) 4. Mechanical and chemical stimuli induce essentially slow motile responses, even though changes in the ionic conditions of the cells and/or their environment can also stimulate the plasma membrane-embedded motor proteins 5, 6. Since OHC motile responses are an essential component of the cochlear amplifier, the qualitative and quantitative analysis of these motile responses at acoustic frequencies (roughly from 20 Hz to 20 kHz in humans) is a very important matter in the field of hearing research 7.
The development of new imaging technology combining high-speed videocameras, LED-based illumination systems, and sophisticated image analysis software now provides the ability to perform reliable qualitative and quantitative studies of the motile response of isolated OHCs to an external alternating electrical field (EAEF) 8. This is a simple and non-invasive technique that circumvents most of the limitations of previous approaches 9-11. Moreover, the LED-based illumination system provides extreme brightness with insignificant thermal effects on the samples and, because of the use of video microscopy, optical resolution is at least 10-fold higher than with conventional light microscopy techniques 12. For instance, with the experimental setup described here, changes in cell length of about 20 nm can be routinely and reliably detected at frequencies of 10 kHz, and this resolution can be further improved at lower frequencies. 
We are confident that this experimental approach will help to extend our understanding of the cellular and molecular mechanisms underlying OHC motility.

A reliable method to investigate outer hair cell (OHC) motile responses, including electromotility, slow motility and bending, is described. OHC motility is elicited by stimulation with an external alternating electrical field, and the method takes advantage of high-speed image recording, LED-based illumination, and last generation image analysis software.

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

Developmental Biology

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after co-culture with cochlear epithelium. The organ of Corti was immobilized on a plastic coverslip by pressing a portion of the Reissner's membrane generated during the dissection. MSCs confined by a glass cylinder migrated toward cochlear epithelium when the cylinder was removed. Their predominant localization was observed in the modiolus of the organ of Corti, aligned in a direction similarly to that of the nerve fibers. However, some MSCs were localized in the limbus area and showed a horizontally elongated shape. In addition, migration into the hair cell area was increased, and the morphology of the MSCs changed to various forms after kanamycin treatment. In conclusion, the results of this study indicate that the coculture of MSCs with cochlear epithelium will be useful for the development of therapeutics via cell transplantation and for studies of cell regeneration that can examine various conditions and factors.

In this study, we present a real-time imaging method using confocal microscopy to observe cells moving toward damaged tissue by ex vivo incubation with the cochlear epithelium containing the organ of Corti.

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after ...

Medicine

Efficient In Vitro Cultivation of Mouse Cochlear Hair Cells

Isolation and Culture of Primary Cochlear Hair Cells from Neonatal Mice

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for hearing, within the osseous labyrinth of the inner ear. Cochlear hair cells consist of two anatomically and functionally distinct types: outer and inner hair cells. Damage to either of them results in hearing loss. Notably, as inner hair cells cannot regenerate, and damage to them is permanent. Hence, in vitro cultivation of primary hair cells is indispensable for investigating the protective or regenerative effects of cochlear hair cells. This study aimed to discover a method for isolating and cultivating mouse hair cells. 
After manual removal of the cochlear lateral wall, the auditory epithelium was meticulously dissected from the cochlear modiolus under a microscope, incubated in a mixture consisting of 0.25% trypsin-EDTA for 10 min at 37 &#176;C, and gently suspended in culture medium using a 200 &#181;L pipette tip. The cell suspension was passed through a cell filter, the filtrate was centrifuged, and cells were cultured in 24-well plates. Hair cells were identified based on their capacity to express a mechanotransduction complex, myosin-VIIa, which is involved in motor tensions, and via selective labeling of F-actin using phalloidin. Cells reached &gt;90% confluence after 4 d in culture. This method can enhance our understanding of the biological characteristics of in vitro cultured hair cells and demonstrate the efficiency of cochlear hair cell cultures, establishing a solid methodological foundation for further auditory research.

Author Spotlight: Advancements in Cultivating Mouse Hair Cells for Auditory Research 

Herein, we present a detailed protocol for isolating and culturing primary cochlear hair cells from mice. Initially, the organ of Corti was dissected from neonatal (aged 3-5 days) murine cochleae under a microscope. Subsequently, cells were enzymatically digested into a single-cell suspension and identified using immunofluorescence after several days in culture.

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for ...

Working with Auditory HEI-OC1 Cells

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Chapters in this video

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis

Optogenetic Stimulation of the Auditory Nerve

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear Surface Preparation in the Adult Mouse

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

Isolation and Culture of Primary Cochlear Hair Cells from Neonatal Mice