Trypsin edta

HEI-OC1 cells, a conditionally immortalized OC cell line, were grown in DMEM (Gibco-BRL, Grand Island, NY, USA) containing 10% FBS (Gibco-BRL, NY, USA) at 33 °C under 10% CO₂. For OC primary cell culture, the cochlea was dissected from SD rats on postnatal day 5. The LW and spiral ganglion neurons (SGNs) were removed, and OCs were enzymatically digested with 0.25% trypsin–EDTA (Gendepot, Barker, TX, USA) in phosphate-buffered saline (PBS) for 5 min at 37 °C. After centrifugation at 3000 rpm for 3 min to remove the supernatant, OC primary cells were plated on cell culture plates and incubated in DMEM (Gibco-BRL, Grand Island, NY, USA) containing 10% FBS (Gibco-BRL, NY, USA) and 0.06 mg/mL penicillin (Sigma-Aldrich, Steinheim, Germany) at 37 °C under 5% CO₂.
Cells were pre-treated with 2-aminoethoxydiphenyl borate (2-APB; Sigma-Aldrich, St. Louis, MO, USA), Ru360 (EMD Biosciences, San Diego, CA, USA), FSK (Sigma-Aldrich, St. Louis, MO, USA), SQ22536 (Sigma-Aldrich), KT5823 (Sigma-Aldrich, St. Louis, MO, USA), diacylglycerol kinase inhibitor (DAGi; Sigma-Aldrich, St. Louis, MO, USA), ATRA (Sigma-Aldrich, St. Louis, MO, USA), H89 (Selleckchem, Shanghai, China), 18 alpha-glycyrrhetinic acid (18α-GA, Sigma-Aldrich, St. Louis, MO, USA), and oleamide (Sigma-Aldrich, St. Louis, MO, USA) for 2 h before CDDP (15 μM, Sigma-Aldrich, St. Louis, MO, USA) application.

The colony formation assay was used to assess cell proliferation. The cells were seeded in 60 Φ (60 × 15 mm) dishes. After incubation for 24 h, cell were subjected to KRG extract treatment for another 48 h. The cells were detached by trypsin (Trypsin-EDTA, GenDEPOT), and then seeded in 6 well plates and grown at 37 °C, in 5% CO₂ incubator for colony formation. After 2 weeks, the cells were stained with crystal violet.