Agilent Technologies Seahorse XFp, User Manual

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6 Agilent Seahorse XFp Glycolytic Rate Assay Kit User Guide
Introduction
Seahorse XF Analyzers directly measure real time extracellular
acidification rate (ECAR) and oxygen consumption rate (OCR)
of cells - indicators of the two major energy-producing
pathways: glycolysis and oxidative phosphorylation. Most cells
possess the ability to switch between these two pathways,
thereby adapting to changes in their environment. To measure
glycolytic rates, Seahorse XF Glycolytic Rate Assay utilizes both
ECAR and OCR measurements to determine the glycolytic
proton efflux rate (glycoPER) of the cells (defined below).
Glucose in cells is converted to pyruvate, and then converted to
lactate in the cytoplasm, or to CO
2
and water in the
mitochondria. The conversion of glucose to lactate results in a
net production and extrusion of protons into the extracellular
medium. (See
Figure 2 on page 8.)


Cells may also use glucose and the other fuels present within
Figure 1 Agilent Seahorse XFp Glycolytic rate assay profile.

Proton efflux from live cells comprises both glycolytic and
mitochondrial-derived acidification. Inhihbition of
mitochondrial function by Rotenone and Antimycin A
(Rot/AA) enables calculation of mitochondrial-associated
acidification. Subtraction of mitochondrial acidification to Total
Proton Efflux Rate results in Glycolytic Proton Efflux Rate.