35 mm glass bottomed dishes

Analysis of mitochondrial membrane potential was done based on a published protocol⁷² . Briefly, transfected cells were grown to confluency on 35 mm glass-bottomed dishes (Corning). Cells were washed in HBSS and incubated in HBSS imaging buffer with 25 nM TMRM for 30 min at 37 °C. Cells were imaged immediately, with TMRM retained in the buffer, using a Leica SP8 confocal microscope and the LAS X software platform, at 37 °C. Images were captured over 2 min, and then 10 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added to dissipate the ΔΨ and control for background. For analysis, an average of the first 10 frames was taken, and then the mitochondria were thresholded and their intensity measured. The average of the final three frames (after CCCP treatment) was subtracted as a control and the intensity of the subtracted image was re-measured.

Cells were seeded onto 35 mm glass-bottomed dishes (Corning). Immediately before imaging, cells were incubated with MitoTracker Green for 30 min at 37 °C, washed three times in HBSS and transferred into imaging media (as above). Imaging was performed using an Olympus IXplore SpinSR system at 37 °C, using 488 and 561 nm laser lines. A z-stack was taken with 10 z-slices, taken every 5 min with 10 time points.

Transfected cells were grown to confluency on 35 mm glass-bottomed dishes (Corning). Cells were washed in HBSS and incubated in HBSS imaging buffer [HBSS supplemented with 5 mM glucose, 10 mM HEPES, 1 mM MgCl₂, 1.26 mM CaCl₂] with 25 nM tetramethylrhodamine (TMRM) for 30 min at 37°C. Cells were imaged immediately, with TMRM retained in the buffer, using a Leica SP8 confocal microscope and LAS X software platform, at 37°C.