In vitro study

In order to clarify the possible cellular and molecular mechanisms after the interaction of hypoxia and gentamicin, we used the HEI-OC1 inner ear cell lines from conditionally immortalized auditory cell lines from transgenic mice (Kalinec et al., 2003). The HEI-OC1 cells, which express characteristic cell markers of organ of Corti sensory cells, have been proved to be a useful in vitro system to study the cellular and molecular mechanisms of drug-induced cochlear cell deaths (Kalinec et al., 2003).

6.2.5.1 Cell culture

The cochlear cells, HEI-OC1 cells, which were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% de-complement FBS (HyClone, Logan, UT, USA). The cells used in this study were maintained at 33 °C under 5 % CO2 in air.

6.2.5.2 Hypoxia of cultured HEI-OC1 cells

HEI-OC1 cells were seeded on culture dish (1 × 10⁶ cells/dish) at 33 °C in 95% air and 5% CO2 before exposure to hypoxia. For the generation of hypoxic condition, cells were

cultured in 95% N2, and 5% CO2 (Anaerobic System PROOX model 110; BioSpherix) condition and incubated at 33 °C within the chamber for 24 h. Cells incubated in hypoxia condition for 0-24 h did not affect cell viability by MTT assay (data not shown).

6.2.5.3 MTT assay

Cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as described previously(Lai et al., 2008). Briefly, after treatment with gentamicin for the indicated time intervals, cultured cells were washed with PBS.

MTT (0.5 mg/ml) was then added to each well and the mixture was incubated at 37^oC for 4 h. Culture medium was then replaced with an equal volume of DMSO to dissolve formazan crystals. After the mixture was shaken at room temperature for 10 min, absorbance of each well was determined at 570 nm using a microplate reader (Bio-Tek, Winooski, VT, USA).

6.2.5.4 Quantification of apoptosis by flow cytometry

Apoptosis was assessed using Annexin V, a protein that binds to phosphatidylserine (PS) residues which exposed on the cell surface of apoptotic cells. Cells were treated with vehicle or gentamicin and cultured in hypoxia for the indicated time intervals. After treatment, cells were washed twice with PBS, and resuspended in staining buffer containing 1 μg/ml PI and 0.025 μg/ml Annexin V-FITC. Double-labeling was performed at room temperature for 10 min in the dark before the flow cytometric analysis. Cells were immediately analyzed using FACScan and the Cellquest program (Becton Dickinson; Lincoln Park, NJ, USA)(Liu et al., 2010).

Quantitative assessment of apoptotic cells was also assessed by cell cycle. Cells were collected by centrifugation and adjusted to 3 × 10⁶ cells/ml. Pre-chilled ethanol was added to 0.5 ml of the cells and incubated at 4°C for 30 min. Ethanol was then removed by centrifugation and DNA of the cells was stained with propidium iodide (PI) [100 μg/ml PI, 0.1% Triton-X, 1 mM EDTA in PBS] in the presence of an equal volume of DNase-free RNase (200 μg/ml) and analyzed immediately by a FACScan and the

Cellquest program (Becton Dickinson).

Quantitative assessment of apoptotic cells was also detected by using TUNEL technique (in situ Cell Death Detection Kit; Roche Applied Science, Mannheim, Germany) according to manufacturer’s instructions. Briefly, cells were incubated with gentamicin in hypoxia for the indicated time intervals. The cells were trypsinized, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton-X-100 in 0.1% sodium citrate. After being washed, the cells were incubated with the reaction mixture for 60 min at 37 °C in the dark. The stained cells were then analyzed using a FACScan and the Cellquest program (Becton Dickinson).

6.2.5.5 Determination of the mitochondrial membrane potential

The mitochondrial membrane potential (ΔΨm) was assessed using a fluorometric probe JC-1 (Calbiochem, CA, USA), with a positive charge of a mitochondrial-specific fluorophore, indicated by a fluorescence emission shift from green (525 nm) to red (610 nm) . Briefly, cells were plated in 6-well culture dishes. After reaching confluence, cells were treated with vehicle or gentamicin. After incubation, cells were stained with JC-1 (5 μg/ml) for 15 min at 37^oC. Samples were analyzed by FACScan using an argon laser (488 nm). Mitochondrial depolarization is specifically indicated by a decrease in the red to green fluorescence intensity ratio and analyzed by a FACScan and the Cellquest program (Becton Dickinson).

6.2.5.6 Measurements of ROS

To detect the level of ROS production, cells were loaded with 10 μM dihydrorhodamine 123 (DHR 123) for 15 min, as described previously (Kim et al., 2008).

The fluorescence intensities were obtained by recording the FITC fluorescence. Cells were collected and analyzed by a FACScan and the Cellquest program (Becton Dickinson).

6.2.5.7 Detection of Ca²⁺ concentrations

Approximately 5 × 10⁵cells/well of HEI-OC1 cells in 12-well plates were incubated with gentamicin in hypoxia for the indicated time intervals to detect changes in Ca²⁺

levels. Cells were harvested and washed twice, and re-suspension in FURA-PE3/AM (3 μM) at 37 °C for 30 min and analyzed by a FACScan and the Cellquest program (Becton Dickinson) (Liu et al., 2010).

6.2.5.8 Western blot analysis

The cellular lysates were prepared as described previously (Chen et al., 2008). Proteins were resolved on SDS-PAGE and transferred to Immobilon polyvinyldifluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The blots were blocked with 5% skim milk for 1 h at room temperature and then probed with antibodies against Bcl-2, Bcl-xl, Bax, Bak, caspase 3, caspase 9, PARP, calpain-1 and calpain-2 (1:1000) for 1 h at room temperature. After three washes, the blots were subsequently incubated with a donkey anti-rabbit peroxidase conjugated secondary antibody (1:1000) for 1 h at room temperature. The blots were visualized by enhanced chemiluminescence using Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY, USA).

6.2.5.9 Determination of caspase activity

The assay is based on the ability of the active enzyme to cleave the chromophore from the enzyme substrate LEHD-pNA (for caspase 9) and Ac-DEVD-pNA (for caspase 3) (Promega; Madison, WI, USA). The cell lysates were prepared and incubated with specific anti-caspase 9 and caspase 3 antibodies. Immunocomplexes were incubated with peptide substrate in assay buffer (100 mM NaCl, 50 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid (HEPES), 10mM dithiothreitol, 1mM EDTA, 10% glycerol, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate (CHAPS), pH 7.4 for 2 h at 37 °C. The release of p-nitroaniline was monitored at 405 nm. Results are represented as the percent change of the activity compared to the untreated control.