For detecting the ROS in dermal fibroblasts I'm using DCFH2-DA dye. By using the fluorescence microscope the ROS levels are not getting detected in the senescence dermal fibroblasts. So, is there any specific protocol for detecting the ROS levels in senescence dermal fibroblasts?
Dear Sravya!
Please You can see in article:
Connective tissue fibroblasts from highly regenerative mammals are refractory to ROS-induced cellular senescence (attached)
Dear Sravya Arikatla,
Nuclear accumulation of 2-hydro-ethidium fluorescence (red) indicative of intra-cellular superoxide (•O2-) generation has been shown by us in GC-1 spg cells and by others in different cell types including senescent fibroblasts. As you are aware, Didydroethidium (DHE) is also known as hydroethidine. In the cytoplasm, DHE manifests blue fluorescence; however, upon oxidation it is converted to ethidium, which then intercalates within DNA, and stains the nucleus a bright fluorescent red.
For your ready reference, I am herewith enclosing our paper (pl. refer to Fig.1) and also providing you a link which may be highly useful:
Victorelli S., Passos J.F. (2019) Reactive Oxygen Species Detection in Senescent Cells. In: Demaria M. (eds) Cellular Senescence. Methods in Molecular Biology, vol 1896. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8931-7_3
https://link.springer.com/protocol/10.1007%2F978-1-4939-8931-7_3#citeas Hope this helps !
Best - Pradyumna
you can use the same protocol and instead of microscopy, you can use facs or plate reader for fluorescence measurement
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