For live MitoSOX and MitoTracker staining and imaging, 2610 4 cells were seeded onto each chamber of tissue culture treated 8-chamber slides (Millicell EZ slides, Millipore, Billerica, MA, USA) and incubated overnight. For MitoSOX staining to detect superoxide generation in the mitochondria, culture media was replaced with 200 mL of staining solution containing 5 mM MitoSOX Red (Invitrogen, Carlsbad, CA, USA) and 5 mg Hoechst 33342 (for nuclear staining, Invitrogen) in HBSS, and cells were incubated at 37uC for 10 minutes. For equilibration, staining solution was replaced with 500 mL pre-warmed HBSS and returned to 37uC for another 10 minutes before imaging. For MitoTracker staining to visualize the mitochondrial network, culture media was replaced with 100 mL of staining solution containing 200 nM MitoTracker Green FM (Invitrogen) and 5 mg Hoechst 33342 in HBSS, and cells were incubated at 37uC for 30 minutes. The staining solution was then replaced with 500 mL pre-warmed HBSS before imaging. Cells were imaged directly in HBSS with a 40x (NA = 0.6) objective on an Olympus IX71 inverted fluorescence microscope equipped with a Coolsnap HQ monochrome camera (Photometrics, Tucson, AZ, USA). For direct comparison of staining intensity, exposure time was kept constant at 500 msec for MitoSOX and at 200 msec for MitoTracker. Staining intensities of MitoSOX and MitoTracker were determined with Image J as pixel intensities, and a minimum of 40 cells each were analyzed.