**3. Isolation of protein complexes from MAMs in cochlea**

### **3.1. Maintenance of mouse cochlear hair cell culture**

#### *3.1.1. Isolation of the organ of Corti*

Decapitate 2 weeks old (CBA/J) mouse at the base of the foramen magnum using scalpel. Briefly rinse the head with 70% ethanol and remove the epidermis using a scalpel blade. Open the cranium along the sagittal suture using a scalpel blade bisect the head equally half and remove the forebrain, cerebellum, and brainstem using blunt dissection. Remove the temporal bones, dip them briefly in 70% ethanol, and transferred into 35 mm dish. Remove the bulla and surrounding tissue from the petrous portion of the temporal bone and identify the conch shaped cochlea and separate it from the vestibular system using forceps. Remove the calcified bony labyrinth of the cochlea carefully removed from basal region to apical end. Spiral ligament and organ of Corti is tightly attached and coiled inside the bony labyrinth. Carefully remove the organ of Corti by securing the spiral ligament at the hook region of the base using forceps and unwinding it as you move apically. Begin at the base and remove the spiral ligament from the organ of Corti using no. 55 fine forceps.

#### *3.1.2. Micro-isolation of hair cells from sensory epithelium of organ of Corti*

The isolation of hair cells from mouse cochlea was described [17]. Remove the organ of Corti at the base of hook region by using two ½ cc insulin syringes with the help of U-100 28G½ needles as forceps. The organ of Corti consist of spiral limbus and sensory epithelium (outer and inner hair cells) cells starting from apex to base of organ of Corti. The sensory epithelium was separated from spiral limbus with help of insulin syringes. The sensory epithelium explant was transferred into fresh Petridis (35 mm) containing 1 ml of DMEM with 10% FBS, ampicillin (10 mg/ml), and 400 μl of each poly-L-ornithine (0.01%) and laminin (50 μg/mL). The Petri dish was incubated at 37°C with 5% CO<sup>2</sup> . After 48 h carefully change the above fresh medium then the adhesive outer and inner hair cells was started multiplication on the Petri dish.
