Adapting high-resolution respirometry for adherent brain endothelial cells reveals stable and reproducible...

High-resolution respirometry enables real-time assessment of mitochondrial respiration but remains challenging in human brain endothelial cells (BECs) due to their low basal respiration and high sensitivity to detachment, which can compromise mitochondrial integrity. We established a standardized and reproducible approach for applying Oroboros O₂k respirometry to BECs. We optimized detachment and suspension conditions to preserve cell viability and enable stable oxygen consumption measurements and performed systematic titration of the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), to define maximal respiratory capacity. Under optimized conditions, oxygen flux measurements were stable with low variability, allowing clear resolution of basal respiration, proton leak, and maximal respiratory capacity. FCCP titration demonstrated maximal respiration was achieved at 2 μM concentration indicating sensitive uncoupler response of BEC mitochondria. We validated the approach across independent experiments, obtaining highly consistent bioenergetic profiles with minimal variability, confirming the reliability of the optimized method. Compared with conventional extracellular flux assays, this approach enables continuous oxygen monitoring and flexible sequential titration, providing improved resolution of mitochondrial parameters. Together, this study establishes a validated and reproducible method for high-resolution Oroboros O₂k respirometry in human BECs, providing a framework for investigating mitochondrial regulation and bioenergetic alterations in BECs in neurological and cerebrovascular disease.