Agilent Technologies Seahorse XFp, User Manual

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Agilent Seahorse XFp Glycolytic Rate Assay Kit
User Guide
Agilent Technologies
4
Frequently Asked Questions
Why does the assay require the use of buffered 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 CO
2
effect the ECAR
measurements?
Acidification of the media by CO
2
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 CO
2
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 CO
2
.
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 CO
2
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