2.7. Analysis of Mitochondrial Function Using Agilent Seahorse XF 96

For analysis of mitochondrial function, IMR-32 and SK-N-BE (2) were plated in 96-well plates at 4 × 10⁵ cells per well. On reaching 90% confluence, cells were treated with 50 μM 4HC for 4 h before mitochondrial respiration was measured using the Seahorse XF 96 Extracellular Flux Analyser and XF Wave Analysis software version 1.4. (Agilent Technologies, Santa Clara, CA, USA) and Seahorse XF 96 Extracellular Flux assay kits. All chemicals were purchased from Agilent Technologies (Santa Clara, USA). Following the 4 h drug treatment, cell culture media was replaced with 200 μl/well DMEM-based XF modified media (free of bicarbonate and phenol red, containing 25 mM glucose and 1 mM pyruvate), and cell plates were incubated for 60 min in a non-CO₂ incubator before sensor calibration was performed and mitochondrial respiration stress test experiments were initiated at a consistent temperature of 37°C. The instrument was programmed for 3 cycles of drug injection, followed by 3 mixing steps and 3 measuring periods (3 min each). The overall experimental time was 110 min. All oxygen consumption rate (OCR) analyses were carried out at least 3 times, with a minimal of 6-12 technical replicates for each treatment. Four different compounds were used to assess mitochondrial OCR: 1.5 μM oligomycin, an inhibitor of mitochondrial membrane adenosine triphosphate (ATP) synthase (F1F0 ATP synthase); 1.0 μM carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP), an ionophore uncoupler of oxidative phosphorylation; 0.3 μM antimycin A, which inhibits the mitochondrial electron transport chain from cytochrome b to cytochrome C1; and 1.0 μM rotenone, an inhibitor of mitochondrial electron transport at nicotinamide adenine dinucleotide: ubiquinone oxidoreductase in complex I.