Microtiter plate reader

HEI-OC1 auditory cells were maintained in high-glucose DMEM containing 10% FBS for characterization. For the experiments described below, HEI-OC1 cells were cultured under the following permissive conditions: 33 °C, 5% CO₂ in DMEM supplemented with 10% FBS. Cells (5 × 10⁴ per well in 24-well plates) were incubated with various concentrations of bucillamine (0.25~4 mM) and cisplatin (20 μM) for 30 h. To determine cell viability, MTT (0.5 mg ml⁻¹ phosphate-buffered saline (PBS)) solution was added to the cell culture media (1/100, v/v) for 4 h and washed with PBS (pH 7.4). Dimethyl sulfoxide was added to each well to solubilize the formazan crystals formed in viable cells. The optical density of each culture well was measured by using a microtiter plate reader (Molecular Devices Co., Sunnyvale, CA, USA) at 590 nm. The optical density of control cells was taken as 100% viability.

Colorimetric assay kits (R&D Systems, Inc., Minneapolis, MN, USA) were used to measure the enzyme activities of caspase-3 and caspase-8 according to the manufacturer's instructions. After 24 h treatment with cisplatin in the presence or absence of bucillamine, the medium was gently removed and discarded, while the cell pellet was lysed by the addition of the lysis buffer. The cell lysates were incubated on ice for 10 min and centrifuged at 10 000 × g for 1 min at 4 °C. The supernatant was then transferred to a new tube to determine caspase activity. Extracts (50 μl) from cells treated with cisplatin in the presence or absence of bucillamine were added to 50 μl of reaction buffer and 5 μl of caspase-3 (DEVD-pNA) or caspase-8 (IETD-pNA) colorimetric substrate. Each mixture was placed in wells of a 96-well plate and incubated for 1 h at 37 °C. Absorbance at 405 nm was measured using a microtiter plate reader (Molecular Devices Co., Sunnyvale, CA, USA). Caspase activity was normalized to micrograms of protein using a Bio-Rad protein assay kit (Bio-Rad Co., Hercules, CA, USA).

Cell proliferation assays were performed using the CellTiter 96 AQueous MTS kit (Promega) and Invitrogen Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye (C10337; Thermo Fisher Scientific) following the manufacturer's instructions. Briefly, a density of 10,000 cells/well was seeded into a 96-well culture plate after lentiviral infection with sh-linc17500, sh-Cks2, or sh-NC. The cells were incubated for specified time points (24, 48, 72, and 96 hours) and then treated with the multicellular tumor spheroids (MTS) solution reagent. Absorbance was measured at 490 nm using a microtiter plate reader (Molecular Devices, San Jose, CA). For the 5-ethynyl-2′-deoxyuridine (EdU) assay, the fluorescein-stained TKE2 cells after 48 hours of cultivation were imaged with a confocal microscope (Carl Zeiss Meditec, Jena, Germany), followed by the use of ImageJ 1.46r (National Institutes of Health, Bethesda, MD) to quantify the proliferating cells. For cell-cycle analysis, 1 × 10⁶ cells were collected and fixed in chilled 70% ethanol for 12 hours at −20°C. The fixed cells were stained with propidium iodide using the BD Cycletest PLUS DNA Reagent Kit (Becton, Dickinson and Company, Franklin Lakes, NJ) according to the manufacturer's instructions. DNA content was analyzed using a BD FACSCalibur Flow Cytometer.