Agilent Technologies Seahorse XFp, User Manual

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22 Agilent Seahorse XFp Glycolytic Rate Assay Kit User Guide
Frequently Asked Questions
Seahorse XF CO
2
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 .
http://seahorseinfo.agilent.com/acton/fs/blocks/showLandingP
age/a/10967/p/p-00ca/t/page/fm/1
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 and all acid sources and does not consider buffering
properties of the assay medium. The Seahorse XF Glycolytic
Rate Assay provides a more precise measurement of
extracellular acidification specifically 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 above. 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.
Questions: Contact Seahorse Technical Support:
Email: Seahorse.support@agilent.com
Ph: 800-227-9770, option 3, option 8