Extracellular flux analyses indicate low ATP yield and require refinement for accurate determination of maximal...

The Agilent Seahorse XF Analyzer has become a central tool for investigating cellular oxidative metabolism. Most studies employ the Seahorse XF Cell Mito Stress Test, which measures the oxygen consumption rate (OCR) under sequential pharmacological modulation of mitochondrial respiration. This assay partitions basal OCR into its functional components and estimates maximal OCR through uncoupler-driven stimulation of respiration, with the resulting value interpreted as the spare respiratory capacity. However, maximal OCR is obtained only after complete inhibition of ATP synthesis by oxidative phosphorylation, and its reliable determination requires appropriate uncoupler titration. Studies have underscored the limitations of this approach. To evaluate its reliability, we examined 97 recent publications reporting 530 Cell Mito Stress Tests. Strikingly, 17% of assays yielded a maximal OCR lower than the basal OCR, raising concerns about the accuracy of the method. To improve maximal OCR determination, we introduce the Cell Stimulation Test, which - analogous to maximal VO₂ assessments in patients - stimulates respiration directly from the basal state across a broad (1-8x) range of uncoupler concentrations. Using the same Seahorse plate, we compared results from both assays. Combined application of the Cell Mito Stress Test and the Cell Stimulation Test enhances the accuracy of maximal OCR assessment and eliminates the need for prior optimization of uncoupler concentration. Our survey further indicates that oxygen consumption not coupled to ATP synthesis constitutes a substantial fraction of basal cellular OCR (median ≈50%), with an unexpectedly large contribution from OCR typically classified as non-mitochondrial. We briefly discuss the implications and limitations of this observation.