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In addition, the day before running the assay, the Seahorse XF Sensor Cartridge was hydrated with sterile water followed by the calibration step (200 L per well of Seahorse XF Calibrant Solution) the day of the experiment in a non-CO 2 37 C incubator

Future work should pair super-resolution and functional imaging with organelle-addressed reporters, localized perturbations (isoform-specific NOX/NOS, ER oxidoreductases, RET modulators) and computational models that capture vectorial flow rather than free diffusion