getting most of ECAR data from a mito-stress test

I have a collaborator that is preparing a publication for submission. They have done seahorse comparing two cell lines (WT and KD for gene X) as part of their study, specifically the mito-stress protocol (oligomycin; FCCP; Rot/AA). They have nice OCR data and can draw several observations from that, but they were also wondering how they can exploit the ECAR data from the mito-stress conditions. They see specifically a difference in the pre-oligomycin injection condition (so basically the + glucose basal condition). I would think they can infer basal glycolytic state from this measurement, but I wanted to check with you if this is acceptable reasoning.  They might be able to do additional Seahorse experiment with these cells, in which case I will suggest they do the Rot/AA – 2-DG combination, but in case that is not doable before submitting the manuscript, they’d like to get the most out of the data they have now. Any suggestions are appreciated.

  • Hi Laura,

    Thank you for contacting Cell Analysis Technical Support.  In general, 
    It is not recommended to use ECAR data from a Cell Mito Stress Test (MST) to assess glycolytic activity for this primary reason: ECAR in the MST is not a direct measurement of glycolysis, but rather a summation of acid (H+) produced from glycolysis and/or the TCA cycle (via CO2). For direct measurement of glycolysis, the XF Glycolytic Rate Assay is recommended.


    ECAR values in the MST represents bulk acidification, including acidification from glycolysis and respiratory CO2. The portion of acidification contributed by COvaries in different cell types and can be evaluated using the Glycolytic Rate Assay, which is able to distinguish between protons (H+) produced from CO2 (respiration) and those produced from glycolysis.

    Qualitative interpretation of the changes in ECAR in MST assays after injection of rotenone and antimycin A may be made in certain circumstances. If the ECAR decreases significantly (i.e. low residual ECAR) in response to rotenone and antimycin A, the ECAR prior to the injection of R/AA is primarily being generated via COproduction from the TCA cycle. If the ECAR remains constant and/or increases in response to rotenone and antimycin A, then the ECAR prior to Rtn/AA treatment is primarily being generated via glycolysis. For detailed information, see Methods of Enzymology article, Vol. 547, pages 332-345.

     Note that any intermediate responses between these two extremes cannot be interpreted accurately from ECAR data alone, and for this reason, the GRA was developed and is recommended. In many cell types, mitochondrial activity that leads to CO2 production is a significant source of extracellular acidification. Unlike bulk acidification assays, the Agilent Seahorse XF Glycolytic Rate Assay is specific for glycolytic acidification. It uses both OCR and ECAR (simultaneously measured by the Seahorse XF analyzer) to measure and then subtract the mitochondrial acidification component, resulting in the glycolytic Proton Efflux Rate (glycoPER), a quantitative and specific measure of glycolytic activity.

     Also note that the Seahorse Glycolytic Proton Efflux Rate  (PER) directly correlates to lactate production. The XF Glycolytic Rate Assay has been designed to correlate with lactate accumulation assays, allowing scientists to cross validate results from different platforms. The key parameter of this assay, glycolytic Proton Efflux Rate (glycoPER), correlates 1:1 with lactate accumulation over time. For more information on the design and validation of the XF Glycolytic Rate Assay, download the GRA white paper.

    Thank you and please let me know if this information helps!


    Courtney Nadeau Watts

    Technical Support Scientist

    Cell Analysis Products

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