2.9. Seahorse Mito Stress Assay

The Seahorse XF96e Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA) was used to generate the bioenergetic profiles of differentiated neuroblastoma SH-SY5Y cell line upon treatments. Live-cell analyses of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Mito Stress test (Agilent). Cells were cultured on a Seahorse XF96 cell culture plate (Agilent, USA) at a density of 5 × 10⁴ cells/well (cell density was optimized to ensure a proportional response of FCCP with cell number) and grown overnight in DMEM 10% of FBS, then differentiated as described above. After complete differentiation, cells were treated as described above. On the day before the Seahorse assay, the cartridge was hydrated and incubated overnight at 37 °C in the absence of CO₂. On the day of the assay, cell medium was replaced with unbuffered DMEM (XF Assay Medium; Agilent Technologies, USA) supplemented with 5 mM glucose and 1 mM sodium pyruvate (Agilent Technologies, USA), and incubated for 1 h at 37 °C in the absence of CO₂. Medium and reagents were adjusted to pH 7.4 on the day of the assay. After four baseline measurements for the oxygen consumption ratio, cells were sequentially challenged with injections of Mito Stress drugs prepared following the manufacturer’s instructions. The final concentrations used for each drug were 1 μM oligomycin (ATP synthase inhibitor), 1 μM FCCP (mitochondrial respiration uncoupler), and 0.5 μM rotenone/antimycin (complex I and III inhibitors). For the normalization in port D, Hoechst 33,342 solution was injected, and at the end of the run, the plate was read using a microplate reader (Infinite Tecan, Männedorf, Switzerland). The data and graphs generated at the end of the Mito Stress assay were extracted using Wave software.