Cellular iron concentration assay

The cellular iron concentration level was detected using a Quantichrom Iron Assay kit (cat. no. DIFE-250, BioAssay Systems). PC12 cells were cultured in 96-well plates and transfected with miR-335 mimic, miR-335 inhibitor, miR-335 NC, or siRNA-FTH1, and stimulated with 6-OHDA for 24 h. Following the manufacturer's protocol, 50 µl of standards or samples containing 10⁶ cells were mixed with 200 µl Quantichrom Working Reagent in a 96-well plate and incubated at room temperature overnight. The optical density (OD) was measured at a wavelength of 590 nm using a microplate reader (BioTek Instruments, Inc.).