The Cell Stimulation Test: an improved protocol for evaluating maximal respiratory rate using the Seahorse Analyzer

Background The Seahorse XF Analyzer developed by Agilent has revolutionized measurement of the oxidative metabolism of cells. Most of these measurements use the Seahorse XF Cell Mito Stress Test, which estimates, among other parameters, the maximal cellular oxygen consumption rate (OCRmax) after respiration is stimulated with an uncoupler. This method has the drawback, however, that the measurement of OCRmax is made after full inhibition of ATP production by mitochondrial oxidative phosphorylation (OXPHOS). Moreover, within this protocol only two additions of uncoupler are possible. This results in a risk that OCRmax is underestimated. As the OCRmax is used to determine the spare respiratory capacity, or ‘power reserve’, of the cells that might be mobilized in the short term, its underestimation might lead to the conclusion that the cells being studied are short of energy, or close to it. Previous studies of cellular bioenergetics reported in the literature have warned about the drawbacks of this procedure for estimating OCRmax. Results Here, we analyzed recent publications that used this protocol and found that in more than 30% of them the OCRmax value presented may be underestimated. We show the results obtained if a “Cell Stimulation Test” is associated to the Cell Mito Stress Test in a single Seahorse experiment. Conclusion The Cell Stimulation Test takes into account experimental constraints and reduces the cellular energy stress under conditions of OCR stimulation by uncoupler. We propose therefore that the Cell Stimulation Test should be associated to the Cell Mito Stress Test to improve characterization of mitochondrial activity within cells.