Oxygen Consumption Rate Analysis

Astrocytes were plated at 7.5 × 10⁴/well in seahorse assay plates and treated with their respective treatments at 37°C and 5% CO₂ and for 24 h. Mitochondrial oxygen consumption rate (OCR) was measured by extracellular flux (XF) assay (Seahorse XFp analyzer, Agilent Technologies, Santa Clara, CA, United States), according to manufacturer’s procedures. Briefly, cells were incubated in a CO₂-free environment for 1 h, and OCR was measured every 3 min for the next 90 min. First, OCR was acquired in basal conditions (20 mM glucose), followed by in the presence of 1.5 μM oligomycin (ATP synthase inhibitor), with 3.5 μM carbonyl cyanide-p-trifluoromethoxy phenylhydrazone (FCCP), and finally, with 0.5 μM rotenone/antimycin A.