Mito stress test

We used the Seahorse Mito Stress test to analyze mitochondrial respiration, following the protocol previously described [23 ]. Briefly, bEnd.3 cells were seeded in XFp > miniplates (7500 cells/well), pretreated for 24 h with vehicle (EtOH; controls) or CoQ10 (5 µM), and treated with Aβ_25–35 (5 µM) for 24 h. Then, cells were incubated in Seahorse XFp > base medium without phenol red (103, 193–100 Seahorse Biosciences) for 60 min at 37 °C before loading into the Seahorse analyzer. Three basal OCR measurements were obtained within the first 20 min. Then, different mitochondrial inhibitors were added [oligomycin, 1 μM; FCCP, 0.3 μM; antimycin A and rotenone, 1 μM]. The inhibitors led us to measure ATP-linked respiration, maximal respiration, and non-mitochondrial respiration, respectively. After different injections, three OCR values were automatically measured by the Seahorse XFp > software Wave 2.6. The OCR levels are represented as pmol of oxygen per minute. Data are presented as the mean ± SEM for each time point normalized to the number of cells per well (n = 3) as previously described [24 (link)].

Primary cultures of rat astrocytes and mixed astrocytes and neurons cultures from hippocampus were prepared from E17 embryonic brains. Astrocyte cultures were seeded onto XFp cell culture miniplate (Seahorse Bioscience) at 5x10⁴ cells/cm² density and grown in Neurobasal 21203, L-glutamine 1 μM, fetal bovine serum 10%, penicillin-streptomycin 50 μg/ml (all reagents from GIBCO Laboratories). In 7 days astrocytes formed a monolayer and allowed for better neuronal attachment to the bottom of the XFp cell culture miniplate. Neurons were seeded onto astrocyte monolayer at 5x10⁴ cells/cm² density and grown in Neurobasal 21203, L-glutamine 1 μM, B-27 supplement, penicillin-streptomycin 50 μg/ml (all reagents from GIBCO Laboratories). For mixed astrocytes and neurons cultures, half of the culture medium volume was exchanged by fresh medium once a week. Astrocytes cultures were used for mitochondrial stress test (MITO STRESS TEST, Seahorse Bioscience) after 17 days in culture, mixed astrocyte and neuron cultures were used after 21–25 days in culture (21–25 days old astrocyte monolayer, 14–19 days old neuronal layer).