4.10. Determination of Intracellular ROS

To determine changes in levels of intracellular ROS production, SH-SY5Y cells were seeded in black 96-well plates and allowed to attach overnight. Thereafter, cells were exposed to BHE and compounds prior to the addition of MPP⁺. After treatment, cells were washed with PBS and stained with 20 µM of 2′,7′-dichlorofluorescin diacetate (DCFDA, Sigma-Aldrich, St Louis, MO, USA) fluorescent dye prepared in un-supplemented for 1 h, and after which cells were washed and treated with dye containing medium replaced with PBS. Fluorescence intensity was read using a microplate reader (BMG Labtech Omega^® POLARStar), and values obtained were expressed as percentage of control.