Orca era ccd camera

Cells were cultured in glass-bottomed microscope dishes and analyzed using an epi-fluorescent microscope with a confocal (quality equivalent) opti-grid device (Nikon TE 2000 microscope equipped with a thermostatted stage and a Hamamatsu Orca-Era CCd camera) and driven by the Volocity 4 operating system (Improvision), which was used for both image data acquisition and analysis [4 (link)]. MMP was measured microscopically with TMRE (tetramethylrhodamine ethyl ester) using Texas Red (excitation 543nm, emission 633nm) filter sets, and the concentration was optimized depending on the cell lines used (0.05–0.5 μM) [4 (link)]. Mitochondrial iron accumulation was measured using RPA (rhodamine B-[(1,10-phenanthrolin-5-yl) aminocarbonyl] benzyl ester) using excitation 564nm, emission 603nm as described in [4 (link)]. Mitochondrial ROS production was determined using mitoSOX Red (Invitrogen, M36008), with excitation 510nm, and emission 580nm as described in [5 (link)]. DFP (ferriprox; 1,2-dimethyl-3-hydroxypyridin-4-one; Apo Pharma) was used as described in [4 (link)–6 (link)].

INS-1E control and shRNA-mNT or shRNA-NAF-1 cells were plated and imaged for mLI accumulation using the probe RPA^{5 (link)}. Mitochondrial ROS production and accumulation were determined by imaging cells with mito-SOX^TM Red (Invitrogen,182M36008). Mito-SOX^TM concentration was optimized for each cell line, on average for all INS-1E cell lines, a concentration of 2.5 μM Mito-SOX^TM was used. All fluorescence images were collected using an Epi-fluorescent microscope with a confocal (quality equivalent) opti-grid device (Nikon TE 2000 microscope equipped with a thermostatic stage and a Hamamatsu Orca-Era CCD camera) and driven by the Volocity 4 operating system (Improvision). The images collected were analyzed by velocity or image-J software programs.