**3.2. Transfection of candidate genes in mouse cochlear hair cells by nucleofector device (Lonza)**

The mouse cochlear hair cell culture is washed with pre-incubated PBS buffer and adds 1 mL of trypsin solution then incubated 37°C for 1 min. and harvest (2.5 × 105) cells. The cells were centrifuged at 300 g for 10 min at room temperature and the supernatant removed and appropriate nucleofector solution (containing 2 μg of plasmid vector or 100 ηM of SiRNA and 100 μL of P4 primary cell 4D-nucleofector X solution) added into the nucleocuvette. Gently tap the nucleocuvette vessels to make sure the samples were premixed and the cover bottom of the cuvette. Place the nucleocuvette vessels and close the lid into retainer of the 4D-nucleofector X unit and select the appropriate program [18]. After completion of the run carefully remove the nucleocuvette vessels and resuspended cells with pre-warmed culture medium. The gene expression or down-regulation will be observed after 4 h transfection to 4 days.
