Ferrostatin 1

Rat retinal precursor (R28) cells (Cat# CVCL_5I35) were provided by Central South University (Changsha, China). R28 cells were maintained in T25 culture flasks with Dulbecco’s modified Eagle’s medium (DMEM) (Procell, Wuhan, China) containing 1 g/L glucose and supplemented with 10% FBS (Gibco, Grand Island, NY, USA) at 37°C with 5% CO₂. Passaging was performed when the cell density reached 80%. Glutamate was obtained from Gibco (Grand Island, NY, USA), SB202190 was purchased from Topscience (Shanghai, China, T2301), and ferrostatin-1 was obtained from Ape Bio (Houston, TX, USA). Glutamate was dissolved in cell culture medium to a concentration of 10 mM. ferrostatin-1 and SB202190 were dissolved in DMSO (MP Biomedicals, Shanghai, China) and diluted in culture medium containing 10 mM Glutamate (Figure 1).

R28 cells were seeded into 12-well culture plates (1 × 10⁵ cells/well) and divided into four groups: the control group, glutamate group (10 mM glutamate), SB202190 group (10 mM glutamate + 25 µM SB202190) and ferrostatin-1 group (glutamate 10 mM + 1 µM ferrostatin-1 [Ape Bio]). Glutamate was dissolved in cell culture medium to a concentration of 10 mM. ferrostatin-1 and SB202190 were dissolved in DMSO (MP Biomedicals) and diluted in culture medium containing 10 mM glutamate. Cells were treated with the respective treatments for 24 hours.
For propidium iodide (PI)/Hoechst 33342 staining, cells were incubated with cell staining buffer, Hoechst staining solution, and PI staining solution at 4°C in the dark for 30 minutes following the manufacturer’s instructions (Beyotime, Shanghai, China). Cells were washed with PBS and observed using a fluorescence microscope (Nikon, Tokyo, Japan). Propidium iodide (PI) indicated dead cells (red fluorescence) and Hoechst 33342 stained all cells (blue fluorescence). The brightness parameters were consistent between the groups during image capture. The photos were analyzed using ImageJ software (Fiji; National Institutes of Health, Bethesda, MD, USA; Schneider et al., 2012). The percentage of cell death was calculated using the formula: cell death rate% = PI-positive cells/Hoechst-positive cells × 100.