Seahorse Mito Stress Test Assay

To validate the effects of mitochondrial inhibitors on metabolic state, we measured rates of astrocytic respiration using an Agilent Seahorse XFp setup and a Seahorse XF Cell Mito Stress Test Kit. Astrocytes were plated and grown to confluency in Seahorse XFp Cell Culture Miniplates, and the Cell Mito Stress assay was performed according to the manufacturer’s protocol. Before the assay, cells were preincubated in Agilent Seahorse XF Base medium containing 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine (pH 7.4). They were subsequently exposed to the commercial reagents of the Cell Mito Stress kit (oligomycin, FCCP, and antimycin A plus rotenone). In the test samples, the commercial mix of antimycin A plus rotenone was replaced with NaCN or rotenone that were used in all functional assays, as specified in figures and figure legends. The acquired oxygen consumption rate values were normalized to the protein content in each well, which was determined using a BCA Assay kit as described in the prior section.