Intracellular reactive oxygen species (ROS) level is measured in

brain mitochondria[31]:

1. Rat brain mitochondrial suspension (0.4 mg/ml) has been

loaded with the cell-permeant probe CM-H2DCFDA (5-(and-

6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate

acetyl ester) (2 μM) for 15 min at 30 °C in dark, and fluorescence is monitored.

2. Using H2DCFDA, an uncharged, cell-permeable fluorescent

probe readily diffuses through the membrane bilayer and gets

hydrolyzed by intracellular esterase to yield H2DCF, which is

trapped inside the cell.

3. Then it is oxidized from the nonfluorescent form to a highly

fluorescent compound dichlorofluorescein by hydrogen peroxide (H2O2) or other low-molecular-weight peroxides produced in the cells.

4. Thus, the fluorescence intensity is proportional to the amount

to the H2O2 produced by the mitochondria.

5. Measure fluorescence through a spectrofluorometer (LS-3B,

PerkinElmer, USA) by using 499 nm as excitation and 520 nm

as emission wavelengths.

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0046594

I hope that following publication will be helpful

https://www.researchgate.net/publication/5544216_Comparison_of_fluorescence-based_techniques_for_the_quantification_of_particle-induced_hydroxyl_radicals

Article Comparison of fluorescence-based techniques for the quantifi...

Thank you both!!!!

Catedrático, Antonio Paz González, contribuição imensurável para ciência! Orgulho de ter sido seu orientado...