Here is an RG discussion that may help:

https://www.researchgate.net/post/How-can-I-distinguish-between-ROS-that-have-mitochondrial-and-non-mitochondrial-origin

Some seem to use DCFDA with and without electron transport chain inhibitors. The use of DCFDA requires cautions as it is easily diffused from the cells as well as could respond to pH and other factors.

Hey Kyoko,

I agree completely with Shin. The normally used DCFDA probe is highly diffusable and as you already said MitoSOX only measures ROS inside mitochondria.

We analyzed extracellular, cytosolic and mitochondrial ROS production during bacterial infection in macrophages and it highly depends on the ROS probe and inhibitor, which you use during your experiments.

As DCFDA, DHE is diffusable and cannot differ between the cytosol and mitochondria. It becomes fluorescent after reaction with superoxide and the binds to any kind of DNA (in mitochondria or the nucleus), which enhances its fluorescence. However, that does not mean by any chance that the ROS you detect with this probe are produced inside mitochondria or the nucleus.

For examples how ROS measurements in different compartments look (especially cytosol and inside mitochondria), please have look at our puplication:

Article Mitochondrial reactive oxygen species enable proinflammatory...

For more information about different ROS probes , scavengers and inhibitors please look at our Review and our Perspective:

Article Functions of ROS in Macrophages and Antimicrobial Immunity

Article Reactive Oxygen Species: Not Omnipresent but Important in Ma...

If you have any further questions please ask.

All the best and stay healthy,

Marc

Respected Dr.Kyoko,

You can estimate ROS from miotochondria by employing several fluorochromes as instructed above. All these fluoroscence imaging methods have limitations and are qualitative.

Are you interested in some particilar ROS?

OH, NO, Superoxide, CO, etc or ONOO ion.

You can have specific probes from Molecular Probes fom Invitrogen. UV light quenches the prrparations as a function of time.

Biochemically, you may perform differential centrifugation and estimate ROS quantitatively colometrically, but it is time consuming.

Are you using a cell free system or tissue?

Hence, time, sensitivity, and specificity, economy, and lab facilities are important considerations in these experiments.

Miotchondria need very special care as these are highly labile organelles and synthesize ROS depending on the metabolic activity of a cell during OP.

I hope it is informative. Good luck with your expts.

Redpectfully submitted

Dean Sharma

Electron Spin Resonance Spectroscopy of the mitochondrial preparations can provide info regarding ROS production in real time however these methods are costly and require skilled researcher. Thanks.

Resectfully submitted,

Dean Sharma

You may like to obtain more info on this issue in recent books.

The Charnoly Body, A Novel Biomarker of Mitochondrial Bioenergetics

Charnolophagy in Health and Disease

Taylor & Francis Science Publishers, CRC Press, Boca Raton, FL, U. S . A. Thanks.

Respectfully submitted,

Dean Sharma

In addition to Digital fluorescence imiging and Confocal microscopy, you may also employ Flow cytometry with DHE stained mitochondrial preps.

Atomic force microscopy and Surface Plasmon Resonance Spectroscopy are other methods to estimate ROS from the mitochondria. TEM/SEM will be time consuming and costly. But choice is your,s and it depends on your passion to discover some thing new and exciting. Thanks.

Respectfully submitted,

Dean Sharma