Xf 24 well culture plates

For assessment of neuronal mitochondrial dysfunction, N2a cells (4 × 10⁴ cells/ml) and primary neurons (8 × 10⁴ cells/ml) were seeded in XF24-well culture plates (Seahorse Bioscience). The cells were washed twice with XF Base Medium supplemented with 2 mM L-glutamine, 10 mM D-glucose and 1 mM sodium pyruvate (pH 7.4) and incubated at 37°C in a non-CO₂ incubator for 1 h. Mitochondrial dysfunction was evaluated using the XF Cell Mito Stress Test Kit (Seahorse Bioscience) according to the manufacturer’s instructions, followed by measurement using an XF24 Extracellular Flux Analyzer (Seahorse Bioscience). The 24-well utility plate was hydrated, treated with 2 µM oligomycin, 2 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and 0.5 µM antimycin A + rotenone, and was then used to calibrate the analyzer. The basal oxygen consumption rate (OCR), ATP production, maximum reserve, and respiratory capacity were calculated as previously described (Dranka et al., 2011 (link)), with averages calculated from 4 wells per condition in each individual experiment. The OCR was normalized to the total protein concentration (OD). After the Seahorse analysis, the plate was centrifuged at 280 × g for 5 min. The media were aspirated, and the plate was washed twice with PBS. The cells were lysed in RIPA buffer. Protein concentrations in cell lysates were determined using a BCA assay kit.