Assay of intracellular reactive oxygen species (ROS)

Intracellular ROS generation in SH-SY5Y cells was detected using the ROS Assay Kit. Typically, SH-SY5Y cells were seeded into a 35-mm glass-bottom culture dish (NEST, China) at a density of 1 × 10⁶ cells/well and cultured overnight in 2 mL DMEM medium with 10% FBS at 37 °C. The medium was replaced with fresh DMEM and 10% FBS with or without Fe²⁺ at a final concentration of 5 mM, followed by addition of 1 µM DRC, 5 µM PM or DPM to a final DRC concentration of 1 µM. Negative control cultures were not exposed to Fe²⁺, free DRC or micelles. The vehicle group was treated with Fe²⁺ and then 0.1 mL DMSO. After 12-h incubation, the medium was replaced with fresh DMEM (2 mL) containing ROS reagent (10 μM), then cells were incubated another 20 min, washed three times with ice-cold PBS and imaged and semi-quantified with fluorescent confocal microscopy (Leica TCS SP5, Germany). The extent of ROS-mediated decomposition of 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) into dichlorofluorescein (DCF) was measured at an excitation wavelength of 488 nm and emission wavelengths of 500 to 540 nm. Three fields of view were randomly chosen for each group before Image-Pro Plus 5.1 software (Media Cybernetics Inc., Rockville, MD, USA) was used for quantitative analysis of ROS (area × intensity).