getting most of ECAR data from a mito-stress test

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 CO₂ varies 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 CO₂ production 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.

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 CO₂ varies 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 CO₂ production 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.