The ECAR and OCR of primary microglia, neurons, and astrocytes were determined using a Seahorse XFe 96 Extracellular Flux Analyzer (Seahorse Bioscience). The ECAR and OCR were measured using the Seahorse XF Glycolysis Stress Test Kit (103020-100, Agilent Technologies) and the Seahorse XF Cell Mito Stress Test Kit (103015-100, Agilent Technologies), and the experimental procedures were performed according to the manufacturer’s instructions. Briefly, primary microglia or astrocytes were seeded into a Seahorse XF 96-well culture microplate (2 × 104 cells per well) overnight (primary cortical neurons were seeded at 1 × 104 cells per well and cultured for 6 days before use), treated with drugs for 24 hours, and then used for ECAR or OCR measurement. Upon measurement, cultured cells were washed twice and maintained in XF assay medium. After baseline measurements, glucose, oligomycin, and 2-deoxyglucose solution or oligomycin, p-trifluoromethoxy carbonyl cyanide phenylhydrazone (1 μM for neurons and astrocytes and 2 μM for microglia), and Rote/AA solution was sequentially injected into the wells of a utility plate at the indicated time points for ECAR or OCR analysis. Data were analyzed using Seahorse XF 96 Wave software, and the results were normalized to cell number and presented as mpH/min for ECAR or pmol/min for OCR.

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