Mitochondrial ROS level was measured by the fluorometric assay, as previously described [26 (link)]. Prepared PC12 cells seeded in 96-well microplates (106 cells/well) were incubated with MitoSOX red reagent (Invitrogen, M36008, USA) at a concentration of 4 μM in the dark at 37°C for 10 min. Cells were then washed twice with warm PBS. The ROS level was identified using a SpectraMax M5e Multi-Mode microplate reader (Molecular Devices, USA).
Protocol detail
Mitochondrial ROS Quantification in PC12 Cells
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Corresponding Organization :
Other organizations : Nanjing Maternity and Child Health Care Hospital, Nanjing Medical University, Nanjing University, Nanjing General Hospital of Nanjing Military Command, Guangdong Province Women and Children Hospital
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Variable analysis
independent variables
- Concentration of MitoSOX red reagent (4 μM)
- Mitochondrial ROS level
- PC12 cell line
- Cell seeding density (10^6 cells/well)
- Incubation time (10 min)
- Incubation temperature (37°C)
- Incubation in the dark
- No explicit mention of positive or negative controls
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