Microscopic Analysis of Mitochondrial Function

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)].

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Karmi O., Holt S.H., Song L., Tamir S., Luo Y., Bai F., Adenwalla A., Darash-Yahana M., Sohn Y.S., Jennings P.A., Azad R.K., Onuchic J.N., Morcos F., Nechushtai R, & Mittler R. (2017). Interactions between mitoNEET and NAF-1 in cells. PLoS ONE, 12(4), e0175796.

Publication 2017

TE 2000 microscope Orca-Era CCd camera mitoSOX Red

Cell lines Cells Confocal microscope Device Dishes Ester Ferriprox Iron Microscope Mitochondrial Orca Rhodamine b Tetramethylrhodamine ethyl ester

Corresponding Organization : University of California, San Diego

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Variable analysis

independent variables

DFP (ferriprox; 1,2-dimethyl-3-hydroxypyridin-4-one; Apo Pharma)

dependent variables

Mitochondrial membrane potential (MMP) measured microscopically with TMRE (tetramethylrhodamine ethyl ester)
Mitochondrial iron accumulation measured using RPA (rhodamine B-[(1,10-phenanthrolin-5-yl) aminocarbonyl] benzyl ester)
Mitochondrial ROS production determined using mitoSOX Red

control variables

Cells were cultured in glass-bottomed microscope dishes
Analysis was performed 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)
Volocity 4 operating system (Improvision) was used for both image data acquisition and analysis

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