2.5.2. Determination of Intracellular ROS by DCFHDA Assay

To investigate the formation of ROS inside SHSY5Y cell line with and without plant extracts, 2,7-dichlorodihydrofluorescein diacetate (DCFHDA) was used [24]. Briefly, SHSY5Y cells were incubated with various concentrations of plant extract (0.25, 0.5, 1, 2, and 4 mg/mL) to induce the antioxidant system in cells using bioactive compounds. After 3 h of incubation, cells were washed with Dulbecco’s phosphate buffer saline (DPBS) and then incubated for 60 min in 10 μM DCFHDA (Molecular Probes, Invitrogen, Basel, Switzerland) in complete medium at 37 °C and 5% CO₂. After the washing step with DPBS, the cells were treated with 200 µM of H₂O₂ for 2 h to undergo intracellular oxidation. Untreated cells were negative control (considered as moderate absorbance) and cells treated with 200 μM H₂O₂ for 1 h were positive control (considered as massive absorbance). Fluorescence intensity was measured using the Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at an excitation wavelength of 485 nm and an emission wavelength of 528 nm.

SHSY5Y cells were incubated separately with different volumes of DMSO (solvent used for solubilizing plant extracts) to check the viability of cells.