Hi,
I am measuring the effect of some compounds on cellular ROS level in HCT116 cell line, and I am wondering is it possible that I detect the ROS increase by MitoSOX, when treating the cells with positive control Rotenone, but I don't detect the overall ROS after the same treatment, measured with DCFH-DA?
The question is, is it possible to detect mitochondrial ROS, which counts for the majority of ROS production in the cell , and at the same time, don't detect any ROS increase using DCFH-DA, which should show the change in overall cellular ROS?
It is not that clear cut. Although DCF-DA is said to measure overall cellular ROS levels, it does not measure everything. It is commonly used as a probe for H2O2. Mitosox, on the other hand, measures mitochondrial superoxide. So it is entirely plausible that you see an increase in mitochondrial superoxide but not in DCF-DA signal. Below is a good review.
Article Measuring reactive oxygen and nitrogen species with fluoresc...
Further discussions are linked below.
https://www.researchgate.net/post/How_can_I_distinguish_between_ROS_that_have_mitochondrial_and_non_mitochondrial_origin
https://www.researchgate.net/post/DCFDA_reaction_with_H2O2
It is not that clear cut. Although DCF-DA is said to measure overall cellular ROS levels, it does not measure everything. It is commonly used as a probe for H2O2. Mitosox, on the other hand, measures mitochondrial superoxide. So it is entirely plausible that you see an increase in mitochondrial superoxide but not in DCF-DA signal. Below is a good review.
Article Measuring reactive oxygen and nitrogen species with fluoresc...
Further discussions are linked below.
https://www.researchgate.net/post/How_can_I_distinguish_between_ROS_that_have_mitochondrial_and_non_mitochondrial_origin
https://www.researchgate.net/post/DCFDA_reaction_with_H2O2
Check this out.
Article Methods for Detection of Mitochondrial and Cellular Reactive...
How about Amplex Red?
Amplex Red can be used to detect H2O2 released from isolated mitochondria.
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