1. After treatment, floating and adherent cells (150.

000 cells per well) were

recovered and incubated in PBS 1X containing 5 μM of Dihydroethidium (DHE)
for 20 min or 10 μM of dichlorodihydrofluorescein diacetate (H 2DCFDA) for 90
min at 37 °C.
2. Dye oxidation, which is visualized by an increase of fluorescence (FL-2 for DHE
and FL-1 for H2DCFDA), was measured using a FACScalibur flow cytometer.
3. With excitation and emission settings at 488 and 585 nm, respectively for DHE
and 488 and 530 nm for H2DCFDA.
4. Positive controls were obtained by incubating cells with 10 mM hydrogen
peroxide.

Dihydroethidium (DHE) was used to detect intracellular superoxide anion

production in HepaRG cells. Since human hepatocytes exhibited stronger
autofluorescence with DHE in our experiments, we used
dichlorodihydrofluorescein diacetate (H2DCFDA) as previously described (Hempel
et al., 1999), even though these two fluorogenic substrates do not measure
exactly the same type of ROS. Indeed, DHE is used to detect the superoxide anion
while H2DCFDA reflects peroxidase activity. These two fluorogenic substrates are
used to detect the oxidative status of the cell, not a type of ROS specifically.

Differential toxic effects of azathioprine, 6-mercaptopurine and 6-thioguanine on human hepatocytes. Toxicology in Vitro Volume 22, Issue 3,
April 2008, Pages 632-642

Cells were loaded with 10 μM dichlorodihydrofluorescein diacetate (DCFH-DA,

Molecular Probes), 2 μM dihydrorhodamine (DHR, Molecular Probes) or 5 μM
previous termdihydroethidiumnext term (DHE, Molecular Probes) by
incubation at 37° for 30 min after melatonin treatments. These probes are
 non-fluorescent cell-permeable compounds; once inside the cell, DCFH-DA is
de-esterified (DCFH) and turn fluorescent upon oxidation (DCF, Rh, Et,
respectively), fluorescence being proportional to ROS production. Analyses
were performed by flow cytometry using FACScan Becton&Dickinson.
Rapid and transient stimulation of intracellular reactive oxygen species by melatonin in normal and tumor leukocytes. Toxicology and Applied
Pharmacology. Volume 239, Issue 1, 15 August 2009, Pages 37-45