Why does the protocol call for adding buffer to the media, will it dampen the ECAR signal?
A low concentration of HEPES (5 mM) has been found to provide consistent buffer capacity values across the time frame of the assay. Although this low concentration of HEPES may reduce the raw ECAR signal slightly, it significantly improves the consistency of the ECAR signal as well as the accuracy of the transformation to PER.
Does acidification of the media by CO2 effect the ECAR measurements?
Acidification of the media by CO2 produced from the TCA cycle can affect ECAR, but its relative contribution varies widely among cell types. By measuring OCR before and after the Rot/AA injection, and using the Buffer Factor (BF) and CO2 contribution factor (CCF), the mitochondrial PER is calculated and subtracted to generate the glycoPER. The Seahorse XF Glycolytic Rate Assay reports the percentage of acidification coming from glycolysis as an easy indicator of whether a substantial amount of acid is coming from CO2.
How do I calculate mitochondrial acidification from mitochondrial oxygen consumption rate? There is a linear correlation between mitochondrial oxygen consumption rate (mitoOCR) and mitochondria-derived acidification (mitoPER), being the CO2 Contribution Factor (CCF), the ratio between mitoPER/mitoOCR, a constant among most cell types. During Glycolytic Rate Assay post-run analysis using Seahorse XF Glycolytic Rate Assay Report Generator, mitochondrial contribution to acidification is calculated from mitoOCR using the pre-determined CCF. However, for cells that are highly oxidative (% PER from glycolysis is <50%) we recommend to reconfirm CCF for the specific cell type using Seahorse XF CO2 Contribution Factor Protocol User Guide. For more details about calculations and derivation of constants, see the Agilent White Paper Improving Quantification of Cellular Glycolytic Rate Using Seahorse XF Technology: XF Glycolytic Rate Assay Whitepaper.
What information does this assay give me that looking at basal ECAR cannot?
Whereas basal ECAR is a good qualitative indicator of glycolysis in most circumstances, it includes acidification of the media from any acid source and does not consider buffering properties of the assay medium. The Seahorse XF Glycolytic Rate Assay provides a more precise measurement of extracellular acidification due to glycolysis by subtracting out mitochondrial sources of acidification, as well as reporting the data in standard units (pmol/min). These features make the Seahorse XF Glycolytic Rate Assay highly comparable to extracellular lactate production measurement assays.
Do I have to calculate the buffer factor (BF) myself?
No- if you are using the recommended formulation. The Buffer Factor has been predetermined for the standard XF Seahorse Glycolytic Rate Assay medium described earlier. If using an assay medium with an alternative composition (different base medium concentrations of substrates), then the buffer factor should be determined empirically in the system following the Seahorse XF Buffer Factor Protocol.
Do I have to use phenol-red free medium? Why?
Phenol red interferes with the pH sensor, causing an apparent pH lower than the actual pH of the assay medium. While this does not affect raw ECAR values, to accurately calculate PER and glycoPER, phenol red must be omitted from the assay media.
More FAQs: Cell Analysis FAQs and Troubleshooting
Keywords: Glycolytic Rate Assay, phenol-red free, Buffer Factor, HEPES, Seahorse, XFe96, XFe24, XFp, FAQ