Cells were plated onto glass coverslips at the confluency of ~40–50% and incubated overnight. The next day 1 µM of DFX was added and cells were incubated overnight. Following the incubation, cells were washed with cold PBS and fixed using 50:50 mix of methanol and acetone (–20°C) for 30 s, blocked in 5% BSA in PBS overnight and incubated with anti-GRP75 antibodies (D-9, Santa Cruz Biotechnology) for 1 hr at room temperature. Cells were washed three times with PBS and incubated with secondary goat anti-rabbit antibody labeled with Alexa Fluor 633 (Invitrogen) in the dark for 1 hr at room temperature. After wash in PBS, cells were mounted onto the glass microscope slides using ProLong Diamond antifade mountant with DAPI (Invitrogen) and imaged using Leica DMi8 confocal microscope after 48 hr.
Imaging with MitoTracker CM-H2Ros was performed using live cells: after incubation with DFX the media was removed and substituted with 200 nM MitoTracker solution in DMEM (no FBS), cells were incubated for 30 min, then media was discarded, and cells were incubated for 5 min in DMEM without serum. Following that, cells were washed with PBS and incubated in 8 µM Hoechst 33422 (Invitrogen) for 10 min, rinsed with PBS, placed in Live Cells Imaging Solution (Molecular Probes) and imaged on Leica DMi8 confocal microscope within 2 hr post staining.
Imaging with MitoTracker CM-H2Ros was performed using live cells: after incubation with DFX the media was removed and substituted with 200 nM MitoTracker solution in DMEM (no FBS), cells were incubated for 30 min, then media was discarded, and cells were incubated for 5 min in DMEM without serum. Following that, cells were washed with PBS and incubated in 8 µM Hoechst 33422 (Invitrogen) for 10 min, rinsed with PBS, placed in Live Cells Imaging Solution (Molecular Probes) and imaged on Leica DMi8 confocal microscope within 2 hr post staining.