Intracellular ROS level was determined using the cell permeant

probe 2′-7′dichlorodihydrofluorescin diacetate (H2DCFDA). Cells

were incubated with 10 μM H2DCFDA in a serum free medium in the

dark at 37 °C for 30 min, collected by trypsinization, washed and

resuspended in an assay buffer (100 mM potassium phosphate, pH

7.4, 2 mM MgCl2). An aliquot was used for protein determination. The

ROS-dependent oxidation of the fluorescent probe (507 nm excitation

and 530 nm emission wavelength) was measured by a Jasco FP6200

spectrofluorimeter.

Article Mitochondrial defect and PGC-1?? dysfunction in parkin-assoc...

I determined the antioxidant activity of different drugs in human gingival fibroblasts (HGF) after the exposure to H2O2.

HGF were grown until confluence in 96-well fluorescence plates (7000 cells/well). At this point, cells were incubated for 0.5 hours with 100 µM H2O2 (H2O2-stimulated group), or in normal conditions (basal group). Then, cells were washed with PBS and treated with an antioxidant molecule or vehicle control overnight added to growth media. Growth medium was removed and cells were stained with 10 µM H2DCF-DA in growth media without fetal calf serum for 0.5 hours at 37˚C. Then, a fluorometer was set to record fluorescence of H2DCF-DA according to the manufacturer's protocol.

Article Identification of Quercitrin as Potential Therapeutic Agent ...

Thank you, Manuel for your brilliant answer

Thank you, Tizian for your brilliant answer