Quantifying Mitochondrial ROS Levels

The level of ROS was assessed as described previously [19 (link)]. Briefly, mitochondrial superoxide was quantified by staining with 5 μM MitoSox red (Thermo Fisher Scientific Inc., Waltham, MA, USA) for 15 minutes. Mitochondria were stained with 100 nM Mitotracker Green FM (Thermo Fisher Scientific Inc.) for 15 minutes. The MitoSox/Mitotracker signal ratio was used to assess the ROS level per mitochondria. All images were collected with an IN Cell Analyzer 6000.

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Nawa N., Hirata K., Kawatani K., Nambara T., Omori S., Banno K., Kokubu C., Takeda J., Nishimura K., Ohtaka M., Nakanishi M., Okuzaki D., Taniguchi H., Arahori H., Wada K., Kitabatake Y, & Ozono K. (2019). Elimination of protein aggregates prevents premature senescence in human trisomy 21 fibroblasts. PLoS ONE, 14(7), e0219592.

Publication 2019

MitoSox red Mitotracker Green FM IN Cell Analyzer 6000

Cell Mitochondria Mitochondrial Mitosox Mitotracker green fm Superoxide

Corresponding Organization : National Institute of Advanced Industrial Science and Technology

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Variable analysis

independent variables

Concentration of MitoSox red (5 μM)

dependent variables

Mitochondrial superoxide level (quantified by MitoSox red staining)
Mitochondrial density (quantified by Mitotracker Green FM staining)
Ratio of MitoSox/Mitotracker signal

control variables

Incubation time for MitoSox red staining (15 minutes)
Incubation time for Mitotracker Green FM staining (15 minutes)
Imaging system (IN Cell Analyzer 6000)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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