Cells were grown to 80% confluency and trypsinized before seeding. Single cells were plated at a density of 2 x 104 onto individual 12mm2 glass coverslips and incubated for 48 h to allow for cells to adhere and begin to grow. Coverslips were stained with 50nM MitoTracker Red CMXRos (Molecular Probes) for 15 min at 37°C, fixed with paraformaldehyde with 0.5% triton-X 100 and quenched with 50mM glycine. To study mitophagy, MOSE cells were fixed in methanol and immunostained with anti-LC3B (Cell Signaling) and with a FITC-conjugated rabbit secondary antibody (Molecular Probes). Coverslips were mounted onto glass slides using Prolong gold antifade mounting medium with DAPI (Molecular Probes) to allow for visualization of the nuclei. Images were captured with a Nikon 80/fluorescent microscope equipped with DAPI, FITC, and TRITC filters using the NIS elements BR 3.0 software and were processed using Adobe Photoshop CS6. DAPI and TRITC images were merged to display localization and spread of mitochondria from the nucleus.

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