What does MitoSOX stain?
MitoSOX™ Red reagent is a novel fluorogenic dye specifically targeted to mitochondria in live cells. Oxidation of MitoSOX™ Red reagent by superoxide produces red fluorescence. The production of superoxide by mitochondria can be visualized in fluorescence microscopy using the MitoSOX™ Red reagent.
How long does MitoSOX last?
If mounted with antifade mountant it can be store at -20 for 2 days, but after that the stain diffuses. It can be counter stained with mitotracker and DAPI to see the changes in intensity of mitochondrial ROS.
How do you use MitoSOX?
Apply 1.0–2.0 mL of 5 μM MitoSOX™ reagent working solution (prepared in step 1.2) to cover cells adhering to coverslip(s). Incubate cells for 10 minutes at 37ГC, protected from light. 1.3 Wash cells. Wash cells gently three times with warm buffer.
Can MitoSOX Red Be Fixed?
Permament incubation of your cells with the staining solution might lead to the observed cell death. I would add the MitoSOX solution, incubate for 15 min at 37 °C, wash one or two times with HBSS and then image your cells in HBSS. As far as I know, MitoSOX is not fixable. Hence, you can’t use MitoSOX on fixed cells.
How do mitochondria produce ROS?
Mitochondrial ROS (mtROS or mROS) are reactive oxygen species (ROS) that are produced by mitochondria. Generation of mitochondrial ROS mainly takes place at the electron transport chain located on the inner mitochondrial membrane during the process of oxidative phosphorylation.
What is mitochondrial superoxide?
Superoxide produced in mitochondria is generated by electrons leaking from the electron transfer system, which is located in the inner membrane of mitochondria. These electrons are then captured by molecular oxygen and become superoxide.
Who discovered Mitophagy?
Mitophagy is the selective degradation of mitochondria by autophagy. It often occurs to defective mitochondria following damage or stress. The process of mitophagy was first described over a hundred years ago by Margaret Reed Lewis and Warren Harmon Lewis.
How does MitoTracker deep red work?
MitoTracker® Deep Red binds thiol‐reactive chloromethyl groups in the mitochondrial membrane and is retained after cell fixation, whereas MitoTracker® Green binds free thiol groups of cysteine residues belonging to mitochondrial proteins and can only be used with non‐fixed cells.
How do you test for mitochondrial ROS?
Investigators have used redox-active probes that, upon oxidation by ROS, yield products exhibiting fluorescence, chemiluminescence, or bioluminescence. Mitochondria-targeted probes can be used to detect ROS generated in mitochondria.
What is MitoTempo?
MitoTempo is a mitochondrial-targeted antioxidant, pre-treatment of cells with MitoTempo protected against hydrogen peroxide-induced cell death. Furthermore MitoTempo significantly reduced mitochondrial superoxide production in cells exposed to preeclampsia plasma by normalising mitochondrial metabolism.
How do you induce ROS?
Another molecule such as COCl2 (an hypoxia mimetic) induces ROS. In all cases, the cytotoxicity must be analyzed in parallel of ROS (DCFH-DA and Hydroethidine). You may try High glucose concentration or VEGF or free fatty acids, It may work.
What does superoxide do in the body?
Superoxide dismutase is an enzyme that helps break down potentially harmful oxygen molecules in cells. This might prevent damage to tissues.
What can mitosox red reagent be used for?
MitoSOX™ Red reagent may be used to distinguish artifacts of isolated mitochondrial preparations from direct measurements of superoxide generated in the mitochondria of live cells. It may also provide a valuable tool in the discovery of agents that modulate oxidative stress in various pathologies. For Research Use Only.
Can you use MitoTracker red and mitosox together?
Since MitoTracker Red and MitoSOX have very similar excitation and emission wavelengths, these two probes cannot be used together. Therefore, we stained MitoSOX separately, using the panel from Table 2. Table 2. Flow cytometry panel for evaluation of mitochondrial ROS in BMDMs.
How is mitosox red indicator used in flow cytometry?
Detection of MitoSOX™ Red indicator by flow cytometry typically uses the FL2 emission channel (585/42 nm). A common application of MitoSOX™ Red indicator is the correlation of superoxide generation with the accumulation of oxidative damage products such as lipid peroxides and modified DNA bases such as 8-oxodG.
What kind of fluorescence is produced by mitosox?
MitoSOX is a mitochondrially targeted fluorescent dye 13 that is oxidized specifically by superoxide to ethidium. The ethidium then intercalates into mitochondrial DNA and thus produces fluorescence proportional to mtROS 39.