Ampicillin

The cells sorted by FAC were cultured in DMEM/F12 medium supplemented with N2 (1%, Invitrogen), B27 (2% dilution, Invitrogen), EGF (20 ng/ml, Sigma), bFGF (10 ng/ml, Sigma), and IGF-1 (50 ng/ml, Sigma), heparin sulfate (50 ng/ml, Sigma) (full medium), and ampicillin (0.1%, Beyotime). For sphere-forming assay, the sorted cells were seeded on TCPS or graphene substrates with 200 cells per well for 5 days. Subsequently, we measured the number and diameter of the formed spheres to evaluate their proliferation capacity. For differentiation assay, the sorted single cells and formed spheres were differentiated separately. The cells or spheres were cultured in a 4-well dish for 10 days, and immunofluorescence staining was then applied to analyze the differentiation of Lgr5+ cells. As described in previous reports, cell aggregation with more than 5 cells were considered a sphere or colony.

The cochleae of P3 wild-type mice were dissected from the inner ear and immersed in bioclean HBSS (Multicell, 311512011) using a stereo microscope. The spiral ganglion, spiral ligament, and stria vascularis of cochleae were removed, and the cochlear basilar membranes were put in dishes that were smeared with Corning^® Cell-Tak™ and then cultured with DMEM-F12 medium added with ampicillin (Beyotime, ST008), N2 Supplement (Stemcell, 07152), and SM1 neuronal supplement (Stemcell, 05711) for 12 h in an incubator at 37°C, 5% CO₂. About 0.01 μm PTV was given to the samples for 12 h, and then, 0.5 mm neomycin with 0.01 μm PTV was given together to the cochleae for another 12 h.

For these experiments, we used pups from the same litter which was reserved for their tails for genotype identification during the experiment.
Kölliker’s organ was micro-dissected in cold 1× Hank’s balanced salt solution (HBSS, Thermo Fisher, 14025076, USA) as described above, transiently transferred to an Eppendorf tube containing DMEM/F12 mixed with 2% ampicillin (ST008, Beyotime, China) and then cultured as previously reported (Chen et al., 2018 (link)). Briefly, Kölliker’s organ was divided into three fragments from apex to base and the fragments were placed in a 24-well plate containing a tissue culture-treated, round glass slide (14 mm diameter, WHB-24-CS, WHB) immersed in DMEM/F12 containing 1% ampicillin, 10% fetal bovine serum (10099-141, Gibco, Australia). Alternatively, for Ca²⁺ imaging experiments with fluo-4 (see below), Kölliker’s organ fragments were placed on a tissue culture-treated glass-bottomed culture dish (801001, NEST, China). In either case, the culture medium was supplemented with 10 μM (Z)-4-hydroxytamoxifen (H7904, Sigma, Germany) to promote Cre recombinase-mediated in vitro excision of the floxed Cx26 alleles. Finally, samples were placed in an incubator (Thermo Scientific Forma Direct Heat CO₂ Incubators) and cultured at 37°C, 5% CO₂ for 12 h (Chen et al., 2018 (link)).