H 1000

Following 24 h incubation coverslips with adherent cochleae were removed from the Maximow slide assemblies, placed in 35 mm petri dishes (Greiner Bio-One 627161) and incubated for 48 h in the presence of 1 ml of cochlear culture medium that had been diluted with DMEM-F12 to reduce the serum concentration to 1.4%, together with 5 μM gentamicin (Sigma G3632) and varying concentrations of berbamine or dTC. Following 48-h incubation, cultures were washed in phosphate buffered saline (PBS), fixed in 3.7% formaldehyde (Sigma F1635) in 0.1 M sodium phosphate buffer pH 7.4, and stained with TRITC-phalloidin (Sigma P1951). Cultures were mounted on glass slides with Vectashield (Vector Laboratories H-1000) and imaged using a Zeiss Axioplan2 microscope. Images were obtained from the middle of the basal coil at a position ~20% along the length of the cochlea, measured from the basal tip. For quantification, the OHCs in these images were counted and averaged across a number of experiments. Image width was 220 μm and was aligned along the length of the cochlea.

Cells were seeded in 6-well plates onto glass slides at 1 × 10⁴ cells/well in 2 ml culture medium. Samples were fixed using methanol-free electron microscopy grade 4% paraformaldehyde in PBS for 15 min at RT, permeabilized for 15 min at RT using 0.15% Trixon X-100 in PBS and blocked for 1 h in 10% BSA (w/v) in PBS. To visualize mitochondria, samples were then incubated with mouse anti-cytochrome c antibody (BD Biosciences 556432, 1:1000) overnight at 4°C and Alexa546-conjugated anti-mouse antibodies (Invitrogen A11003, 1:500) for 1 h at RT. Nuclei were stained by incubation with DAPI (Invitrogen, D1306, 1:1000) for 5 min right after Alexa546 incubation. Samples were mounted in mounting medium (Vectashield, H1000) and observed using a confocal microscope (Zeiss LSM Meta710, 63 × / 1.4 objective). Mitochondria fragmentation and Parkin-to-mitochondria translocation were assessed visually and quantified as percentage of control. The extend of mitochondrial fragmentation was judged based on comparison to untreated control cells with mitochondrial networks scored as fragmented if most mitochondria in a cell did no longer exhibit an elongated phenotype. All experiments were performed independently in triplicates.