Agilent Technologies Seahorse XFp User manual

Agilent Technologies
Agilent Seahorse XFp
Glycolytic Rate Assay Kit
User Guide
Kit 103346-100

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Manual Part Number
103346-400
Kit Part Number
103346-100
Edition
Second edition, May 2019
Revision B0
Printed in USA
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Agilent Seahorse XFp Glycolytic Rate Assay Kit User Guide 3
Contents
Introduction
Assay Background 5
Glossary 9
Kit Information
Kit Contents 11
Kit Shipping and Storage 11
Additional Required Items 12
Assay Workflow
Day Prior to Assay 14
Day of Assay 14
Running the Assay 18
Analysis Using the Agilent Seahorse Glycolytic Rate Assay Report
Generator 19
Frequently Asked Questions

4 Agilent Seahorse XFp Glycolytic Rate Assay Kit User Guide

5
Agilent Seahorse XFp Glycolytic Rate Assay Kit
User Guide
Agilent Technologies
1
Introduction
Assay Background 5
Glossary 9
Assay Background
For use with an Agilent Seahorse XFp Extracellular Flux
Analyzer.
The Agilent Seahorse XFp Glycolytic Rate Assay is an accurate
and reliable method for measuring glycolysis in cells. Together
with a Seahorse XFp Analyzer, the Seahorse XFp Glycolytic
Rate Assay provides accurate measurements of glycolytic rates
for basal conditions and compensatory glycolysis following
mitochondrial inhibition (see Figure 1 on page 6). The
calculated rates accounts for contribution of CO2to
extracellular acidification derived from mitochondrial/TCA
cycle activity, and are directly comparable to lactate
accumulation data.

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 CO2and 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.

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 CO2can
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/CO2to
extracellular acidification is calculated and used to subtract the
CO2contributing acidification from the total Proton Efflux Rate
(PER). (For a detailed explanation, please read the Agilent
Seahorse XF CO2Contribution 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 CO2- 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.

8 Agilent Seahorse XFp Glycolytic Rate Assay Kit User Guide
Introduction
Figure 2 Principle of the Agilent Seahorse XFp Glycolytic Rate Assay.
Energy is produced by two pathways in the cell - glycolysis,
and mitochondrial respiration. In the breakdown of glucose to
lactate during glycolysis, protons are extruded into the
extracellular media which is detected by the XFp Analyzer as
ECAR. Additionally, mitochondrial TCA activity produces CO2,
which hydrates and acidifies the media. By inhibiting
respiration (OCR) during the assay with the complex I and
complex III mitochondrial inhibitors (Rot/AA), the rate of
proton efflux from respiration can be calculated and removed
from the total proton efflux rate, giving the glycolytic proton
efflux rate (glycoPER). To confirm pathway specificity, 2-DG, an
inhbitor of glycolysis is injected to stop glycolytic acidfication.

Introduction
Agilent Seahorse XFp Glycolytic Rate Assay Kit User Guide 9
Glossary
• Glycolysis: In the context of the Seahorse XFp Glycolytic
Rate Assay, the process of converting glucose to lactate.
• Buffer factor (BF): Buffer capacity of the measurement
system, comprising the assay medium and XF assay
conditions (instrument, sensor, labware).
• Proton efflux rate (PER): The number of protons exported
by cells into the assay medium over time, expressed as
pmol/min.
• Glycolytic proton efflux rate (glycoPER): The Proton
Efflux rate derived from glycolysis (discounting the effect of
CO2-dependent acidification). This measurement is highly
correlated with the extracellular lactate production rate.
• Compensatory glycolysis: The rate of glycolysis in cells
following the addition of mitochondrial inhibitors,
effectively inhibiting oxidative phosphorylation, and driving
compensatory changes in the cell to use glycolysis to meet
the cells' energy demands.
• Post-2-DG acidification: This value includes other sources
of extracellular acidification that are not attributed to
glycolysis or mitochondrial TCA activity as well as any
residual glycolysis not fully inhibited by 2-DG. It is
measured after the addition of 2-DG in the Glycolytic Rate
Assay workflow.
•Inducedassay:Assay workflow that includes an acute
injection of an experimental compound prior to injection of
XF Glycolytic Rate Assay compounds. This workflow allows
for real-time quantification of in situ glycolysis activation or
repression.

10 Agilent Seahorse XFp Glycolytic Rate Assay Kit User Guide
Introduction