H9c2 cells were trypsinized with a 0.25% trypsin-EDTA solution (Hyclone) and centrifuged at 1,000 rpm for 5 min at room temperature. For measurement of total ROS, the pellet was resuspended in culture medium, and cells were incubated with 2′,7′-dichlorofluorescein diacetate (DCF-DA; 20 μM, Alexis Biochemicals) for 30 min at 37 °C. To quantify mitoROS, cells (2×105 cells/well) in the 96-well culture plate were incubated with the mitochondrial superoxide-sensitive fluorescent dye MitoSOX Red (1 μM, Invitrogen, Carlsbad CA) for 20 min at 37 °C [42 (link)]. Fluorescence intensity was measured using a Spectramax M3 plate reader (Molecular Devices, Sunnyvale, CA) at excitation/emission of 485 nm/530 nm for DCF-DA and 510 nm/580 nm for MitoSOX Red. Antioxidant effects of XJB were evaluated in cells treated with 10 μM DMNQ or 1 μM antimycin A.
Mitosox red
Manufactured by Molecular Devices
Sourced in United States
MitoSOX Red is a fluorogenic dye that is selectively targeted to the mitochondria of live cells. It is commonly used as a probe for the detection of superoxide in the mitochondria.
Automatically generated - may contain errors
Lab products found in correlation
Mitosox red, by Thermo Fisher Scientific (5 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Dcf da, by Molecular Devices (1 mentions)
2 7 dichlorofluorescein diacetate dcf da, by Alexis Biochemicals (1 mentions)
Spectramax m3 plate reader, by Molecular Devices (1 mentions)
Dihydroethidium dhe, by Thermo Fisher Scientific (1 mentions)
Spectramax i3 plate reader, by Molecular Devices (1 mentions)
Spectramax e5, by Molecular Devices (1 mentions)
Imagexpress micro confocal microscope, by Molecular Devices (1 mentions)
4 protocols using mitosox red
1
Quantifying Cellular Oxidative Stress
2
Quantifying Intracellular and Mitochondrial ROS
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Intracellular and mitochondrial ROS production were measured by the superoxide indicator dihydroethidium (DHE) (Thermo Fisher Scientific) and the mitochondrial superoxide indicator MitoSOX Red (Thermo Fisher Scientific). Briefly, cells were infected with Lgy parasites or treated with poly I:C for 8 hours, after which cells were labelled with 5μM DHE or 5 μM MitoSOX Red in PBS with 5 mM glucose (Gibco) for 20 min at 37°C. After incubation, cells were washed, and fluorescence was measured at 518/606 nm (MitoSOX Red) and 510/590 nm (DHE) using the Spectramax i3 plate reader (Molecular Devices). Measures were normalized to total protein concentration per well using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) following manufacturer’s instructions.
3
Measuring Mitochondrial Oxidative Stress
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Mitochondrial superoxide generation was monitored by MitoSOX Red (Invitrogen) fluorescence. SH-SY5Y cells (50,000 cells/well) were seeded in to black walled, clear bottomed 96-well plate. Cells were incubated with inhibitors (40 nM, 370 nM, 3000 nM) for 1 h before adding 35 μM 6-OHDA for 5 h. Cells were stained with 2.5 μM MitoSOX Red for 25 minutes under growth conditions. The cells were washed twice in Hank's Buffer Salt Solution (HBSS), and placed in pre-warmed HBSS for imagining. MitoSOX Red fluorescence was detected by exciting the fluorophore at 510 nm and monitoring the emission at 580 nm on a SpectraMax e5 plate reader (Molecular Devices). Mitochondrial superoxide was normalized to cell abundance by staining the cells with Hoechst 33342 (excitation: 350 nm; emission: 450 nm).
4
Mitochondrial Superoxide Measurement
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Cells were plated in 24-well plates and loaded with 5 μM MitoSOX Red (M36008, Invitrogen, USA) in phenol-free DMEM for 10 min at 37 °C with 5% CO2 and washed with PBS. MitoSOX Red fluorescence intensity was determined at 510 nm excitation and 580 nm emission under an ImageXpress Microconfocal microscope (Molecular Devices, USA).
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