Need a protocol to define ROS generation in different cellular compartments such as cytoplasm and mitochondria.
Also need suggestion of any particular inhibitor of ROS for respective compartments
Dear Saswat,
I would go for plamid (roGFP) introduction targeted to mitochondria and cytoplasm. You can check this:
For mitochondria:
https://www.addgene.org/49437/
https://www.addgene.org/64977/
https://www.addgene.org/49436/
for cytoplasm:
https://www.addgene.org/49435/
regards
For mitochondrial ROS you can measure by mitosox red and for scavenging you have different ROS scavengers like tempol for ROS and mitotempol for mitochondrial specific ROS
Dear Saswat, you can stain your samples with DCF-DA (ROS-sensitive dye) and TMRE (conventional mitochondrial dye) simultaneously and check for presence or absence of yellow fluorescence. I think it is the easiest and the cheapest way.
For quenching ROS you should keep in mind the purposes of you research. Because antioxoidant could be water-soluble (NAC, Trolox) or hydrophobe (Vit E). The first scavenge ROS like O2- or H2O2, the second - stop lipid peroxidation in membranes. In some cases this difference could be very important.
There is a number of mitochondrial-targeted antioxidants. The most used are MitoTEMPO and SkQ1.
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