Dichloro-Fluorescein Diacetate (DCFH-DA) Assay

The ability of the HI extract to protect against the oxidative stress in T47D-KBluc, A549, and HGF cells was monitored using the reactive oxygen species (ROS) sensitive 2,7 dichloro-fluorescein diacetate (DCFH-DA) dye. After a 24 h treatment with non-toxic HI extract concentrations, the cells were washed with PBS and further loaded with 50 μM DCFH-DA in Hanks' balanced salt solution (HBSS) for 2 h. Following incubation, the excess of DCFH-DA was washed, and the cells were exposed to 250 μM H2O2 for 2 h. The conversion of DCFH-DA to the fluorescent compound dichlorofluorescein (DCF) was measured using Synergy 2 multi-mode microplate reader at λexcitation = 485/20, λemission = 528/20. The potency of HI extracts, to mitigate the induction of oxidative stress after H2O2 treatment, was compared to N-Acetyl Cysteine (NAC) treatment (20 mM solution).
