Glutamate glu

To test the anti-ferroptotic properties of ADA-409-052, PC-12 cells were exposed for 24 h to either 0.25 µM RSL3 (Selleck Chemicals, TX, USA) or 20 mM glutamate (Glu, Sigma) with or without ADA-409-052 at following concentrations: 2.5, 5, 10, and 20 µM. Minocycline (Sigma) was used in parallel at the same concentrations. The compounds’ efficacy in cell protection was measured as cell viability 24 h post-treatment using the resazurin assay. In brief, cells were incubated for 2 h at 37 °C with 10 µM resazurin in HBSS. The absorbance was measured at 485 nm. Additionally, ADA-409-052’s effect on intracellular GSH levels was measured. We thus exposed PC-12 cells to 20 mM glutamate with or without 5 µM ADA-409-052. After 24 h of exposure, GSH was extracted from the collected cell pellet with 5% sulfosalicylic acid and the supernatant neutralized with 3 M Tris. The Amplite Rapid Fluorimetric Glutathione GSH/GSSG Ratio Assay Kit (AAT Bioquest, Sunnyvale, CA, USA) was used to measure the GSH concentration according manufacturer’s instructions. All results were expressed as percentage of mean fluorescence from untreated control cells.

Glutamate (Glu) and 17β-estradiol (E2) were purchased from Sigma Aldrich (St. Louis, MO, USA and Madison, WI, USA, respectively).

Leadacetate and chloroplatinic acid were purchased from Sinopharm Chemical Reagent company (Beijing, China). Graphene oxide nanocomposites solution (2 mg/mL) was purchased from Xianfeng Corporation (Nanjing, China). Nafion solution (20%), ascorbic acid (AA), uric acid (UA), dopamine (DA), 5-hydroxytryptamine (5-HT), dihydroxyphenylacetic acid (Dopac) and glutamate (Glu) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Saline (0.9% NaCl) was purchased from the ShuangHe Corporation (Beijing, China).