Agilent Technologies Seahorse XFp, Руководство пользователя

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Introduction
Agilent Seahorse XFp Glycolytic Rate Assay Kit User Guide 7
the cell or in the assay medium for energy production through
mitochondrial respiration. Mitochondrial-derived CO
2
can
partially hydrate in the extracellular medium, resulting in
additional extracellular acidification beyond that contributed
by glycolysis. By also measuring the amount of oxygen
consumed by the cell, the contribution of mitochondria/CO
2
to
extracellular acidification is calculated and used to subtract the
CO
2
contributing acidification from the total Proton Efflux Rate
(PER). (For a detailed explanation, please read the Agilent
Seahorse XF CO
2
Contribution Factor Protocol User Guide ).
The resulting value, glycoPER, is the rate of protons extruded
into the extracellular medium during glycolysis. This assay
allows for real-time measurements of changes in glycolysis rates
that may go undetected in long-term lactate accumulation
assays.
The assay workflow is as follows: first, cells are incubated in the
Seahorse XF Glycolytic Rate Assay Medium containing
substrates such as glucose, glutamine and pyruvate, as well as
HEPES buffer, and basal rates are recorded over 3 measurement
periods. Next, Rot/AA (inhibitors of mitochondrial electron
transport chain) are injected to inhibit mitochondrial oxygen
consumption (and therefore CO
2
- derived protons). The second
injection is 2-deoxy-D-glucose (2-DG), a glucose analog which
inhibits glycolysis through competitive binding of glucose
hexokinase, the first enzyme in the glycolytic pathway. The
resulting decrease in PER provides qualitative confirmation
that the PER produced prior to the injection is primarily due to
glycolysis.