Mitochondrial Superoxide Imaging with MitoSOX

Mitochondrial O₂⁻ production was measured using MitoSOX Red indicator (Life Technologies) as described (Tronchere et al., 2017 (link)). Briefly, cells were loaded with MitoSOX (2 µm) for 30min, washed in PBS, PFA-fixed, mounted on glass slide using MOWIOL and imaged on a Zeiss LSM780 confocal microscope. Frozen heart cryosections were hydrated in PBS and incubated with MitoSOX (5 µm) for 30 min at 37°C in a humidified chamber, quickly washed and imaged on a Zeiss LSM780 confocal microscope. The MitoSOX fluorescence intensity was quantified using Image J software on thresholded images.

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Boal F., Cinato M., Timotin A., Münzberg H., Qualls-Creekmore E., Kramar S., Loi H., Roncalli J., Keita S., Tronchere H, & Kunduzova O. (2022). Galanin Regulates Myocardial Mitochondrial ROS Homeostasis and Hypertrophic Remodeling Through GalR2. Frontiers in Pharmacology, 13, 869179.

Publication 2022

MitoSOX Red indicator LSM780 confocal microscope

Cells Confocal microscope Cryosections Fluorescence Frozen Heart Mitochondrial Mitosox

Corresponding Organization :

Other organizations : Université Toulouse III - Paul Sabatier, Inserm, Pennington Biomedical Research Center, Louisiana State University System, Université de Toulouse

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Variable analysis

independent variables

Mitochondrial O2- production

dependent variables

MitoSOX fluorescence intensity

control variables

Cell loading with MitoSOX (2 µm) for 30 min
Washing in PBS
PFA-fixing
Mounting on glass slide using MOWIOL
Hydration of frozen heart cryosections in PBS
Incubation with MitoSOX (5 µm) for 30 min at 37°C in a humidified chamber
Quick washing of frozen heart cryosections

controls

No positive or negative controls were explicitly mentioned in the provided information.

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