Seahorse

CHAPTER SIXTEEN
Analysis and Interpretation of

Microplate-Based Oxygen
Consumption and pH Data
Ajit S. Divakaruni*,2, Alexander Paradyse*, David A. Ferrick,
Anne N. Murphy*,1, Martin Jastroch{,1
*Department of Pharmacology, University of California, San Diego, California, USA
Seahorse Bioscience, Billerica, Massachusetts, USA

{
Institute for Diabetes and Obesity, Helmholtz Zentrum Munchen, German Research Center for
Environmental Health, Neuherberg, Germany
1
Denotes equal contribution
2
Corresponding author: e-mail address: adivakaruni@ucsd.edu
Contents
1. Introduction
2. The Oxygen Consumption Rate
2.1 Cellular respiration
2.2 Data analysis and normalization
2.3 Using permeabilized cells to localize bioenergetic changes observed
with intact cells
3. The Extracellular Acidification Rate
3.1 Medium acidification associated with glycolytic turnover
3.2 Medium acidification not attributable to glycolytic turnover
3.3 Characterizing cellular ATP demand with respiration and extracellular
acidification
Summary
Acknowledgments
References
310
311
311
320
326
330
332
341
345
347
347
347
Abstract
Breakthrough technologies to measure cellular oxygen consumption and proton efflux
are reigniting the study of cellular energetics by increasing the scope and pace with
which discoveries are made. As we learn the variation in metabolism between cell types
is large, it is helpful to continually provide additional perspectives and update our
roadmap for data interpretation. In that spirit, this chapter provides the following for
those conducting microplate-based oxygen consumption experiments: (i) a description
of the standard parameters for measuring respiration in intact cells, (ii) a framework for
data analysis and normalization, and (iii) examples of measuring respiration in permeabilized cells to follow up results observed with intact cells. Additionally, rate-based
Methods in Enzymology, Volume 547

ISSN 0076-6879
http://dx.doi.org/10.1016/B978-0-12-801415-8.00016-3
2014 Elsevier Inc.

All rights reserved.
309
310
Ajit S. Divakaruni et al.
measurements of extracellular pH are increasingly used as a qualitative indicator of glycolytic flux. As a resource to help interpret these measurements, this chapter also provides a detailed accounting of proton production during glucose oxidation in the
context of plate-based assays.
1. INTRODUCTION
Classical approaches to measure oxygen consumption such as Clarktype electrodes are limiting when measuring respiration in intact cells, as
they require large amounts of cells that must be amenable to stirring and prolonged suspension. The recent development of fluorescence- or
phosphorescence-based oxygen sensors, however, allows bioenergetic measurements in adherent cell monolayers (Dmitriev & Papkovsky, 2012;
Gerencser et al., 2009). These multi-well, plate-based methods reduce
the sample material required by orders of magnitude and allow simultaneous
measurements of multiple experimental conditions. They also expand the
number of cell types that can be studied, and physiological relevance is
enhanced by maintaining interactions with the extracellular matrix and preserving mitochondrial populations that can be lost upon trypsinization (e.g.,
those in processes of primary neurons). Since the introduction of these
methods, there has been a tremendous increase in our understanding of
how mitochondrial function changes in response to drugs and drug candidates, exogenously added substrates, cell signaling events, and targeted
changes in gene expression.
The availability of these technologies has tracked with a resurgent interest in cellular metabolism. In particular, altered flux through specific metabolic pathways has received attention in oncogenesis (Caro et al., 2012;
Hensley, Wasti, & DeBerardinis, 2013; Maddocks et al., 2013; MarinValencia et al., 2012), cardiovascular disease (Fillmore & Lopaschuk,
2013), insulin resistance (Gimenez-Cassina et al., 2014; Muoio et al.,
2012; Newgard et al., 2009), neurodegeneration (Gimenez-Cassina et al.,
2012), immune function (MacIver, Michalek, & Rathmell, 2013;
Pollizzi & Powell, 2014; Tannahill et al., 2013; van der Windt et al.,
2012; Wang et al., 2011), and adaptation to hypoxia (Fan et al., 2013;
Metallo et al., 2012). Longstanding observations about aerobic glycolysis
are not only being revisited in cancer (Israelsen et al., 2013; Lunt &
Vander Heiden, 2011; Onodera, Nam, & Bissell, 2014), but also increasingly
examined in new contexts including immunology (Chang et al., 2013;
Gubser et al., 2013; Macintyre et al., 2014; Sukumar et al., 2013) and glucose
Microplate-Based Oxygen Consumption and pH Data
311
handling by terminally differentiated cells (Keuper et al., 2014; Teperino

et al., 2012). This appreciation for the broad scope of cellular bioenergetics
has also translated into increased drug development efforts for targeting
energy metabolism in cancer (Deberardinis, 2014; Tennant, Duran, &
Gottlieb, 2010), neurodegeneration (Cunnane et al., 2011), and other conditions that are not traditionally considered metabolic diseases.
As our collective experience with different cell types grows within the
community of mitochondrial research, it is increasingly clear that there is
immense variation in metabolism between cell types. Certainly the foundational principles of bioenergetics do not change between experimental systems (Nicholls & Ferguson, 2013). However, new findings have already
challenged some of our textbook-based perceptions of energy metabolism
and suggest a one-size-fits-all approach may be limiting. Therefore, it is
important to continually refine our roadmap for interpreting cellular respiration data to better understand metabolism between different cell types as
well as within individual models in response to genetic or pharmacologic
interventions.
This chapter is presented to provide an additional perspective on the
interpretation of respiration and extracellular pH data. The work was conducted using Seahorse Extracellular Flux (XF) Analyzers, but the concepts
are applicable for many plate-based methods. Other valuable reviews of
XF technology are available in the context of general bioenergetics to characterize mitochondrial dysfunction (Brand & Nicholls, 2011), oxidative stress
(Dranka et al., 2011), mitochondrial quality control (Hill et al., 2012), and
technical considerations for measuring mitochondrial function in diabetes
(Perry, Kane, Lanza, & Neufer, 2013). This chapter addresses issues that
we believe warrant further consideration. Topics addressed include different
bioenergetic profiles between proliferating and differentiated cells, data analysis and normalization, the use of permeabilized cells to reveal the
source(s) of metabolic dysfunction, and the biochemical framework for
why glucose oxidation acidifies the extracellular medium. The following
chapter assumes the reader has some familiarity with bioenergetic terminology and cellular respiration measurements, as well as a working knowledge
of oxidative phosphorylation, glycolysis, and acidbase chemistry.
2. THE OXYGEN CONSUMPTION RATE

2.1. Cellular respiration
Measurement of cellular respiration is a powerful experimental tool to assess
energy metabolism because of the coupling between ATP synthesis and
312
oxygen consumption during oxidative phosphorylation. Electrons are

harvested from oxidizable substrates such as glucose, amino acids, and fatty
acids and transported through the electron transport chain in a series of exergonic (energetically downhill) reactions that ultimately reduce molecular
oxygen to water. This electron transfer is coupled to the endergonic (uphill)
pumping of protons from the matrix to the intermembrane space to generate
a protonmotive force (p) across the inner membrane. Various cellular processes consume this potential, including the FoF1 ATP synthase, which dissipates p to drive the rotary catalysis of the enzyme to produce ATP.
As the terminal reaction in the electron transport chain, oxygen consumption is both a measurement for the flux of electrons through the respiratory chain (supply of p) as well as an indirect measurement of the
processes that consume p (demand). The most broadly informative measurements involve monitoring cellular respiration in response to a series of
chemical effectors that partition respiration rates into distinct modules, as
shown in Fig. 16.1. The experiment follows a longstanding convention
by bioenergeticists to measure mitochondrial function. It is grounded in
Mitchells chemiosmotic theory of oxidative phosphorylation (Mitchell,
1961) and verified by decades of work with both cells and mitochondria
using platinum-based oxygen electrodes. Its adaptation to the Seahorse
XF Analyzer has been previously covered in-depth (Brand & Nicholls,
2011; Hill et al., 2012). A description of the measurements, their interpretation, and what processes control each oxygen consumption rate (OCR)
are presented here and summarized in Fig. 16.1.
2.1.1 Basal respiration
After removing the contribution from non-mitochondrial oxygen consumption (Section 2.1.6), the basal (or endogenous) rate of respiration is
mostly composed of two processes: respiration used to drive ATP synthesis
and that which is associated with proton leak pathways. The proportion of
basal respiration that is used to drive ATP synthesis can be easily estimated by
addition of the ATP synthase inhibitor oligomycin. As mitochondrial ATP
production slows, so too will the respiration used to drive it. The rate of
ATP-linked respiration can therefore be approximated by the rate of oxygen
consumption sensitive to oligomycin. Although this value is often an underestimate (Section 2.1.3), the error is generally thought to be small (Affourtit &
Brand, 2009). Particularly in electrically excitable cells such as primary neurons, some proportion of the basal respiration rate can also reflect the transport
of calcium or other ions across the inner membrane (Duchen, 1992). This
Figure 16.1 Defining, calculating, and interpreting assay parameters for plate-based oxygen consumption measurements. C2C12 myoblasts
were maintained according to ATCC specifications and plated at 1 104 cells/well in XF96 microplates. After 2 days, cells were washed and
assayed in unbuffered DMEM (Sigma #D5030) supplemented with 10 mM glucose and 1 mM pyruvate. Where indicated, oligomycin (2 g/mL
final), FCCP (800 nM final), and rotenone (1 M final) with antimycin A (2 M final) were acutely added via the injector port. Data are from a
single biological replicate and presented as mean standard error of the mean (S.E.M.) of six wells.
314
consumption of the membrane potential stimulates increased activity of the

electron transport chain to maintain the membrane potential, and will present
as oligomycin-insensitive respiration (see Fig. 16.4A as an example).
2.1.2 ATP-linked respiration
Three processes can control the rate of ATP-linked respiration: (i) ATP utilization (energy demand), (ii) ATP synthesis, and (iii) substrate supply and
oxidation.
(i) ATP utilizationIn many cell types, cellular ATP demand will set the
rate. ATP-linked respiration will therefore proportionally change based
on activity of energy-requiring processes like macromolecule biosynthesis, cell motility, proteasome activity, or specialized cell functions
like membrane repolarization during synaptic neurotransmission or
muscle activity. Specific inhibitors of ATP-consuming processes can
be used to create a hierarchy of which pathways compose the overall
rate of respiration (Birket et al., 2011; Buttgereit & Brand, 1995).
The amount of respiratory inhibition seen upon inhibitor addition
can be attributable to a priori activity of a given ATP-dependent process.
(ii) ATP synthesisA decrease in ATP-linked respiration can also reflect a
reduced ability to consume the membrane potential and synthesize
ATP. Impaired activity of the ATP synthase, the adenine nucleotide
translocase (which exchanges ADP for newly synthesized ATP in
the matrix), or the phosphate transporter (which imports phosphate
and a proton into the matrix for ADP phosphorylation) can slow
the rate of ATP synthesis.
(iii) Substrate supply and oxidationA decreased rate observed upon drug
treatment or genetic modification can also indicate reductions in the
ability to generate a membrane potential and fuel ATP synthesis.
A profound defect in substrate supply (transport at the plasma or mitochondrial membrane), substrate oxidation (including rates of glycolysis
or TCA cycle activity), or respiratory chain activity (particularly complex I) could limit the ability of the cell to meet its energy demand.
Discriminating between these three rate-controlling processes can be
achieved by additional oxygen consumption measurements. FCCPstimulated rates of respiration (Section 2.1.4) can reveal changes in substrate
supply and oxidation, and offering substrates and ADP to permeabilized cells
(Divakaruni, Rogers, & Murphy, 2014) or isolated mitochondria (Rogers
et al., 2011) can quantify activity of the proteins responsible for synthesizing
ATP (permeabilized cells are discussed in Section 2.3).
315
In some nutrient-sensing cells types, most notably pancreatic -cells

(Affourtit, Jastroch, & Brand, 2011; MacDonald, Joseph, & Rorsman,
2005) and presumably hypothalamic neurons (Ainscow, Mirshamsi, Tang,
Ashford, & Rutter, 2002; Parton et al., 2007), substrate supply will primarily
set the rate of respiration rather than ATP demand. In the case of the -cell,
this allows the cell to enhance oxidative glucose catabolism in response to glucose supply, increasing the ATP/ADP ratio and potentiating
insulin exocytosis. By and large, regulation of basal respiration by nutrient
supply is thought to be the exception rather than the rule (Ainscow &
Brand, 1999).
Nonetheless, the importance of medium composition in the study of
bioenergetics should be appreciated. In models using both primary and
immortalized cell types, exogenously added pyruvate can increase the rate
of basal respiration, perhaps suggesting that glycolytic provision of pyruvate
may be rate-controlling in some cell types (Choi, Gerencser, & Nicholls,
2009; Diers, Broniowska, Chang, Hill, & Hogg, 2014). Moreover, the
Crabtree effect, a hallmark of cancer in which respiration is suppressed by
elevated concentrations of glucose, is apparent in several cell culture models
and is not exclusive to cancer cells (Palorini, Simonetto, Cirulli, &
Chiaradonna, 2013; Redman, Brookes, & Karcz, 2013; Robinson,
Dinsdale, Macfarlane, & Cain, 2012; Sweet et al., 2009; Fig. 16.4B).
2.1.3 Proton leak-linked respiration
The protonmotive force generated during substrate oxidation is not used
exclusively to drive ATP synthesis. Some protons leak back across the
mitochondrial inner membrane, constantly consuming the membrane
potential and stimulating activity of the respiratory chain to maintain it
( Jastroch, Divakaruni, Mookerjee, Treberg, & Brand, 2010). This respiration uncouples respiratory chain activity from ATP production, and is the
reason why some mitochondrial respiration (and substrate oxidation) persists
in the presence of oligomycin.
The molecular nature of constitutive proton leak is not fully understood,
and only a small fraction is attributable to leak through the lipid bilayer itself
(Brookes, Rolfe, & Brand, 1997). A larger proportion is attributable to the
abundance (but not activity) of the adenine nucleotide translocase and speculatively occurs at the proteinlipid interface (Brand et al., 2005). Mitochondrial anion carrier proteins such as uncoupling proteins (UCPs) can
acutely and chronically regulate proton leak, the most notable example
being UCP1 in brown adipose tissue (Nicholls, 2001). A growing body
316
of research continues to reinforce an important role for proton leak in physiology. In specific cell types, plasticity in the coupling of nutrient oxidation
to ATP synthesis can regulate heat production for thermoregulation, mitochondrial reactive oxygen species production, glucose-stimulated insulin
secretion, and disengaging carbon flux from ATP demand. The physiological functions of UCPs 13 are reviewed in Divakaruni and Brand (2011).
Measurements of the respiration associated with proton leak are valuable
for identifying molecular mechanisms that alter proton conductance across
the mitochondrial inner membrane, but caution should be exercised during
data interpretation. Proton leak-linked respiration is steeply dependent on
the protonmotive force used to drive it. As such, changes in p will
result in altered proton leak-dependent respiration without changing the
proton conductance of the inner membrane. When matched changes
are observed in both proton leak-associated respiration and uncouplerstimulated respiration, there may be no difference in the membrane conductance itself, as changes in the membrane potential likely explain the result
(Divakaruni & Brand, 2011; Keipert & Jastroch, 2014).
The standard assay described in Fig. 16.1 underestimates the actual rate of
ATP-linked respiration because of the dependence of proton leak on the
mitochondrial membrane potential. Oligomycin will slightly hyperpolarize
the mitochondrial membrane and overestimate the endogenous rate of proton leak-linked respiration, but the error is estimated to be generally less than
10% (Affourtit & Brand, 2009). To unequivocally identify whether altered
proton conductance is driving the changes seen in respiration, measurements
of membrane potential must be conducted. If a mechanism inherent to the
mitochondria is causative, measurements can be taken using isolated mitochondria or permeabilized cells (Affourtit, Quinlan, & Brand, 2012). If
the cytoplasmic environment is required, quantitative assessment of
mitochondrial membrane potential in whole cells is possible but technically
challenging (Gerencser et al., 2012; Nicholls & Ward, 2000).
2.1.4 Maximal respiration
Basal rates of respiration do not adequately reflect the ability of cellular respiration to respond to an increased energy demand. This is particularly true
for electrically excitable cells such as neurons or myocytes, which temporarily face periods of high ATP demand to re-establish ion gradients that drive
neurotransmission or contraction. As such, estimating the maximal capacity
of substrate oxidation can be tremendously valuable to reveal mechanisms by
which genetic modifications or pharmacologic compounds affect cellular
317
metabolism. Moreover, for those designing respirometry experiments to

examine drug effects on mitochondrial substrate oxidation, maximal rates
of respiration offer large dynamic windows for concentrationresponse
curves.
Protonophore-stimulated respiration is a good estimate of the maximum respiratory capacity of the cell, and thus can measure the maximum
capacity for mitochondrial substrate oxidation. Compounds such as
FCCP, DNP, or Bam15 will shuttle protons across the inner membrane,
dissipating p and causing maximal activity of the electron transport chain
to maintain the protonmotive force (Nicholls, 2008).These molecules are
therefore referred to as protonophores or uncouplers, as allowing proton
re-entry into the matrix uncouples respiratory chain activity from ATP
synthesis.
It is absolutely essential that the concentration of uncoupler used to stimulate maximal respiration be optimized by titration. Excess protonophore
can cause a precipitous decrease in the OCR. The precise cause is unclear
and likely due to several parameters, but may be somewhat attributable to a
reduced driving force for substrate uptake into the matrix (Clerc & Polster,
2012). Respiratory substrates such as pyruvate and glutamate are anions, so
reductions in the pH gradient and membrane potential by protonophore
addition could limit substrate provision to the respiratory chain. Because
optimal concentrations can vary between experimental groups (e.g., genetic
modifications, drug treatments, medium composition, etc.), the injector
ports can be used for sequential additions of uncoupler to ensure a maximal
rate is achieved (Figs. 16.2 and 16.4). Use of less potent uncouplers such as
DNP or Bam15 may, to a degree, circumvent this problem ( Jastroch,
Keipert, & Perocchi, 2014; Kenwood et al., 2014).
The rate of protonophore-stimulated respiration in an intact cell may not
always reveal the maximum respiratory capacity. If a cell is highly dependent
on mitochondrial ATP production, prior addition of oligomycin may limit
ATP-dependent processes that supply oxidative pathways, such as maintenance of the plasma membrane sodium gradient that helps drive glucose
uptake. Moreover, substrate oxidation could be robust, but limitations in
substrate transport across either the plasma or mitochondrial membrane
may limit the rate of uncoupler-stimulated respiration. If a change in maximal respiration is observed, measurements of mitochondrial content and
cristae density (Section 2.2.3), substrate-specific analysis in permeabilized
cells (Section 2.3), and respiratory chain complex activity assays can diagnose
the mechanism of action responsible for the effect.
Figure 16.2 Oxygen consumption profiles for proliferating and terminally differentiated cells. As in Fig. 16.1, arrows indicate sequential additions of oligomycin (2 g/mL
final), FCCP (given for each panel), and rotenone with antimycin A (1 and 2 M final,
respectively). When four arrows are shown, two sequential pulses of FCCP were injected.
(A) Top left: Cortical astrocytes were isolated and maintained according to published
protocols (Kim & Magrane, 2011). Cells were plated at 1.25 104 cells/well in XF96 plates
and grown for 4 days. On the day of the assay, medium was replaced with aCSF (Clerc &
Polster, 2012) supplemented with 10 mM glucose and 1 mM pyruvate. [FCCP] 600 nM
followed by 300 nM (900 nM final). Top center: C2C12 myoblasts were maintained
according to ATCC and plated in XF24 plates at 2 104 cells/well. After 2 days, medium
was exchanged to unbuffered DMEM supplemented with 8 mM glucose, 3 mM glutamine, and 3 mM pyruvate. [FCCP] 800 nM followed by 200 nM (1 M final). Top right:
A549 lung adenocarcinoma cells were maintained in DMEM/F-12 medium (Gibco; other
specifications according to ATCC) and plated in XF96 plates at 1.5 104 cells/well. After
2 days, medium was exchanged to unbuffered DMEM supplemented with 8 mM glucose, 3 mM glutamine, and 3 mM pyruvate. [FCCP] 600 nM. (B) Bottom left: Cortical
neurons were isolated and maintained as previously described (Kushnareva, Wiley,
Ward, Andreyev, & Murphy, 2005) and plated at 1.8 104 cells/well in XF96 plates.
After 14 days, cells were assayed in aCSF supplemented with 10 mM glucose and
1 mM pyruvate. [FCCP] 300 nM followed by 150 nM (450 nM final). Bottom center:
C2C12 myotubes were cultured and plated as in (A), but after 2 days, medium was
exchanged for similar growth medium but 10% (v/v) FBS was omitted and 2% (v/v)
horse serum was added. After 6 days, cells were assayed in unbuffered DMEM supplemented with 8 mM glucose, 3 mM glutamine, and 3 mM pyruvate. [FCCP]
800 nM followed by 400 nM (1.2 M final). Bottom right: White adipocytes were isolated
and differentiated according to Keipert and Jastroch (2014). Cells were plated at 1 104
cells/well in XF96 plates and assayed in unbuffered DMEM supplemented with 25 mM
glucose, 10 mM pyruvate, and 0.3% (w/v) BSA. [FCCP] 1 M final. Rotenone and
antimycin A were each added at final concentrations of 2.5 M. All data are from a single
biological replicate and presented as mean S.E.M. from a minimum of five wells.
319
2.1.5 Reserve capacity/Spare respiratory capacity

The reserve capacity (Hill et al., 2012), or spare respiratory capacity
(Nicholls, 2009; terms used here interchangeably), is defined as the difference between basal and protonophore-stimulated respiration. Broadly, it
has been suggested that a reserve capacity exists so a cell can appropriately
respond to an increased ATP demand and withstand periods of stress. If
the experimental test is simply to measure the oxidative capacity of a cell,
maximal respiration is often the more appropriate metric because it is purely
a function of substrate supply and oxidation. Moreover, as spare respiratory
capacity is dependent on multiple parameters, it does not explicitly identify
molecular mechanisms of action. However, within a given physiological
context such as oxidative stress (Dranka et al., 2011; Dranka, Hill, &
Darley-Usmar, 2010) or ion homeostasis in synaptoneurosomes (Choi
et al., 2009), the reserve capacity is useful in synthesizing effects on several
respiratory parameters into a single value. Like all bioenergetic measurements, it is important to view the reserve capacity within the context of a
particular model system. Just as increased proton leak-associated respiration
can reflect either mitochondrial damage (Dranka et al., 2011) or a normal
physiological response (Divakaruni & Brand, 2011), the lack of spare respiratory capacity does not per se indicate dysfunction.
Figure 16.2 shows a panel of oxygen consumption experiments from a
range of cell types. As a metric to measure the spare respiratory capacity with
an internally normalized parameter, the ratio of maximal respiration to
ATP-linked respiration was calculated (basal respiration can also be used).
Maximal respiration in cortical astrocytes, C2C12 myoblasts, and A549 lung
carcinoma cells (Fig. 16.2A, upper row) all display less than a threefold
increase over ATP-linked respiration (2.0-, 2.9-, and 2.0-fold, respectively).
Conversely, the ratios in cortical neurons, differentiated C2C12 myotubes,
and white adipocytes (Fig. 16.2B, lower row) all exhibit increases of fivefold
or higher (5.4-, 5.0-, and 6.3-fold, respectively). The results highlight the
need to interpret spare respiratory capacity within a cell-specific context.
Although cortical astrocytes or C2C12 myoblasts do not possess the same
reserve capacity as cortical neurons or C2C12 myotubes, this likely reveals
more about cell physiology rather than energetic dysfunction. The cells with
relatively low reserve capacities in Fig. 16.2 are all proliferative and likely
exhibit proportionally higher ATP-linked respiration to meet the biosynthetic demands of cell replication. Those cells with higher spare respiratory
capacities are all post-mitotic. They have relatively low resting demands but
are characterized by mitochondrial biogenesis during terminal differentiation
320
and the capacity to execute energetically demanding, cell-specific functions.

Similar results are observed upon differentiation of primary human myocytes
(Nisr & Affourtit, 2014).
2.1.6 Non-mitochondrial respiration
To correct for oxygen consumption from non-mitochondrial oxidases,
inhibitors of respiratory complex I (rotenone) and III (antimycin A) are
added at the end of an experiment to stop mitochondrial electron transfer.
In some hematopoetic cells such as monocytes and leukocytes, nonmitochondrial respiration can be a significant contributor to the overall rate
(Kramer, Ravi, Chacko, Johnson, & Darley-Usmar, 2014), presumably due
to activity of flavin-linked NAD(P)H oxidases (Bedard & Krause, 2007).
The rate has been shown to increase in response to redox cycling compounds (Dranka et al., 2010), and robust activity can be characterized when
enzymes are specifically activated and inhibited (e.g., the use of phorbol
esters and flavin inhibitors in blood cells; Chacko et al., 2013). The recent
development of new microplates to study three-dimensional model systems
shows, remarkably, that HCT116 cells grown in two-dimensional cultures
exhibit relatively high rates of non-mitochondrial respiration that are absent
when grown in spheroids (http://www.seahorsebio.com/learning/
webinars/details.php?wID63), an observation that can be reproduced in
our laboratory. Determining the extent to which this is driven by oxidase
activity, as opposed to being a result of instrumentation, could prove valuable in the study of non-mitochondrial oxygen consumption.
2.2. Data analysis and normalization

2.2.1 Data analysis
Along with a summary of what can compose and control each rate, Fig. 16.1
presents sample calculations for a given data set. It is essential that metrics be
calculated from experiments that exhibit relatively stable rates, particularly
for measurements of basal respiration. We suggest taking the minimum or
last measurement in the presence of oligomycin because its bioavailability
can often result in a time-dependent effect, particularly for multicellular
structures like spheroids or pancreatic islets (Wikstrom et al., 2012).The
same is true for the calculation of non-mitochondrial respiration, but in
two-dimensional cultures the time dependence should be minimal.
Protonophore-stimulated rates in many cells types are relatively stable,
though a slight decrease in rate is often seen over time. We suggest taking
the maximum rate obtained during an experiment irrespective of the
321
uncoupler concentration required to stimulate that rate, though this depends

somewhat on the experimental question being asked. Most importantly, the
researcher should be aware of the complicating factors when measuring
maximal respiration (Section 2.1.4). Reductions in substrate transport or
availability could cause a time-dependent rundown in the uncouplerstimulated rate of respiration that does not reflect the maximum capacity
for cellular substrate oxidation. In cells with a high oxidative capacity, this
could also be caused by depletion of oxygen in the well or insufficient recovery of the fluorophore to its baseline oxygen tension (Gerencser et al., 2009).
The user should always check the raw [O2] values (termed level in Seahorse XF/Wave software) if concerned about any measurement. Possible
solutions to accurately measure high rates of oxygen consumption include
lowering cell density, increasing the period between measurements
(mix and wait commands), and decreasing the measurement time
(the minimum for the XF Analyzer is 2 min).
2.2.2 Data presentation
Because respiratory rates are a function of several parameters, details such as
plating conditions, days of growth, medium composition (both growth and
assay), and concentrations of mitochondrial effectors used should be
reported to best facilitate comparison between different laboratories. This
is also why it is often best to report raw rates whenever possible and avoid
using area under the curve (AUC). This calculation does not allow comparisons to historical data, laboratories using different equipment (e.g., comparing rates from the Seahorse XF Analyzer with platinum-based electrodes;
Rogers et al., 2011), or even those using the same platform with different
timing schemes. Moreover, from a purely empirical perspective, timedependent rate changes from compound bioavailability or altered substrate
transport by protonophores can affect an AUC analysis in ways that are likely
not biologically meaningful.
2.2.3 Considerations for normalizing data
A singular advantage of plate-based methods is the comparison of multiple
experimental groups, but the value of these experiments can be lost with
large variability within the microplate. Care should be taken to plate cells
evenly between wells, and we highly recommend the use of a multichannel
pipette and mixing cells in a reservoir multiple times before plating them in a
row or column. Experimental groups should be chosen so they do not overlap with the method of plating (i.e., if plating cells one column at a time,
322
consider setting up experimental groups in rows or half-columns on either

side of the plate). Moreover, long-term cultures are particularly susceptible
to effects of temperature and humidity at the periphery of the microplate.
Attention must therefore be given to whether the arrangement of experimental groups on the plate can explain observed differences in the OCR.
Care should be taken to place seeded XF microplates towards the back of
the incubator to minimize changes in temperature and humidity from frequent opening. The researcher should also consider placing medium or PBS
in the outer wells for long-term cultures, seeding cells in only the inner
60 wells of a 96-well plate, if sufficiently concerned about effects from
the microplate edges (Lundholt, 2003).
If a change in rate is observed between groups, consideration must also be
given to whether a given intervention (genetic modification, chronic drug
treatment, etc.) has changed the total cell number, mitochondrial mass
within the cell, viability, or adherence. Particularly when cells are grown
or differentiated over several days in multi-well plates, changes in respiration
due to altered cell number can be misattributed without appropriate normalization. Several methods of normalization are possible, some of which are
listed here.
Total cellular protein: Among the easiest and most inexpensive methods for
normalization is to lyse cells and measure the total protein in each well of the
XF plate. Although a relative comparison may be all that is needed, a standard curve should always be conducted with known protein amounts to
ensure the reading exists in the pseudolinear range of the signal. Like all protein assays, it is critical that the lysis buffer used not interfere with the
detection method.
While this method is often appropriate for a cursory analysis of whether
cell number has changed, it requires the assumption that the total protein per
cell has not changed with a given intervention. In some instances, targeted
genetic manipulation of protein expression can result in a remarkable change
in cell size and total protein in a given cell (Wiley et al., 2013). Moreover,
normalization to total protein content will minimize changes in mitochondrial biogenesis that may drive a particular respiratory phenotype, as the
changes in mitochondrial protein may be masked within the larger context
of total cellular protein. For these reasons, it is often important to normalize
respiratory rates to the total number of cells in a given well.
Cell number: The most sophisticated (and expensive) methods for normalization to cell number are to use automated, high throughput imaging
devices designed specifically for such purposes (e.g., Celigo S (Nexcelom)
323
or automated widefield microscopy as in Affourtit et al., 2011). In their

absence, a more cost-effective approach is to use a nuclear stain such as
Hoechst or picoGreen as in Fig. 16.3. This requires a fluorescent plate reader
that can be programmed to accommodate the unique dimensions of the XF
plate. It is best practice to generate a standard curve of signal to known cell
numbers to understand the sensitivity of the assay.
It is also possible to assess levels of cytoplasmic enzymes (e.g., lactate
dehydrogenase (LDH)) with linked assays after their release upon cell
permeabilization. Again, a standard curve should be generated, and the user
should be mindful that experimental interventions could alter enzyme
expression without matched changes in cell number. If running a standard
respiratory assay that concludes with addition of respiratory chain inhibitors,
it is not advised to use a live cell assay that relies upon loading of an esterified
fluorescent dye. Cellular de-energization upon addition of rotenone and
antimycin A can limit viability and adherence, compromising the ability
of the cell to load and de-esterify the dye. As such, independent, matched
plates should be set aside to use this particular approach.
Mitochondrial content: An altered maximum rate of respiration relative to a
matched control could indicate a change in mitochondrial mass or volume
relative to cell number. Several approaches can address this issue, and the
assessment likely requires parallel plates of cells, usually with larger surface
Figure 16.3 Use of a DNA stain to quantify muscle fibers in the microplate well. Primary
FDB muscle fibers were prepared essentially according to published methods (Schuh,
Jackson, Khairallah, Ward, & Spangenburg, 2012) and seeded into XF96 plates. Fibers
in each well were counted by light microscopy, subsequently lysed in buffer containing
proteinase K, and transferred to a microtitre plate. PicoGreen (Life Technologies) was
diluted 1:200 in TE buffer, and 100 L of this solution was added to each well. After
a 5 min incubation, an end-point measurement was taken using 485/520 filters.
324
area, treated analogously to the XF plate. Ultimately, the most rigorous and
informative method to assess mitochondrial content is morphometric analysis with electron micrographs (Frey, Perkins, & Ellisman, 2006). Other
approaches include measuring mitochondrial:nuclear DNA ratios
(Marine, Krager, Aykin-Burns, & Macmillan-Crow, 2014) or quantifying
immunoblots of multiple electron transport chain proteins normalized to
a cytoplasmic protein (Wiley et al., 2013). It is also possible to compare
activities of mitochondrial enzymes, such as citrate synthase or cytochrome
oxidase, to cell number. Here again, the caveats about altering enzyme content without matched changes in mitochondrial mass or volume must be
considered.
2.2.4 Using the baseline feature in Seahorse software
The XF/Wave software allows the user to scale rates to the fractional change
relative to a defined measurement, so-called baselining. It can be useful to
minimize the variability during plating and visualize relative changes from an
acute injection. Reporting scaled rates in the absence of absolute rates, however, does not allow comparison of respiratory rates between experiments
from different laboratories. Additionally, absolute rates can often be diagnostic for whether an assay was conducted correctly. If groups of cells have been
differentially treated in any manner prior to the assay, using the baseline feature can reduce the amount of information gained during an assay and, in
some cases, lead to misinterpretation of the data. Drug treatment, genetic
modification, or altered medium composition may not only change the cell
number but perhaps also the energy demand of the cell or the capacity to
meet that demand. With any type of pretreatment, it should not be assumed
that the basal or oligomycin-insensitive rate should remain the same
between experimental groups.
Figure 16.4 shows three examples of how information can be lost when
using the baseline feature to scale respiratory data to the initial rate. It demonstrates how the basal rate of respiration can change in response to
increased ATP demand (Fig. 16.4A), medium composition (Fig. 16.4B),
or lowered capacity for cellular substrate oxidation (Fig. 16.4C).
Figure 16.4A shows cortical neurons pre-treated with veratridine, which
opens a plasma membrane Na+ channel and increases the ATP demand from
enhanced activity of the Na+/K+ ATPase. As seen previously in synaptoneurosomes (Choi et al., 2009), the treatment also causes a slight
increase in respiration insensitive to oligomycin and a mild decrease in
uncoupler-stimulated respiration. This is perhaps due to, respectively,
325
Figure 16.4 Information lost upon normalizing to the initial oxygen consumption rate.
Oxygen consumption data is presented without (AC, upper panels) and with (DF,
lower panels) use of the baseline function in XF/Wave software. (A and D) Primary cortical neurons were maintained, plated, and assayed as in Fig. 16.2 with the following
exceptions. Where indicated, wells were treated with 4 M veratridine for 15 min prior
to the initial measurement. [FCCP] sequential additions of 200 nM (400 nM final).
(B and E) The hypothalamic astrocyte cell line CLU201 (formerly rHypoE-6 Embryonic
Rat Hypothalamic Cell Line R6) was maintained according to the manufacturer's protocol (CELLutions). Cells were plated at 4 104 cells/well in XF96 plates one day prior to
measurements. Cells were offered 5 mM pyruvate in unbuffered DMEM and, where
indicated, 15 mM glucose. [oligomycin] 2 g/mL. (C) Primary cortical neurons were
maintained, plated, and assayed as in Fig. 16.2D. Where indicated, 10 nM myxothiazol
(myxo.) was added 15 min prior to the initial measurement. [FCCP] sequential
additions of 200 nM (400 nM final). All data are from a single biological replicate and
presented as mean S.E.M. from a minimum of five wells.
increased Ca2+ cycling and mitochondrial Ca2+ overload. When normalized

to the rate immediately prior to oligomycin injection, the increase in ATP
demand upon treatment is lost, and a slight change in maximal respiration
from veratridine addition is exacerbated to mistakenly suggest severe mitochondrial dysfunction (Fig. 16.4D).
Similarly, CLU201 hypothalamic astrocytes were offered medium with
5 mM pyruvate and either no glucose or 15 mM glucose. In these cells, a
pronounced Crabtree effect (the suppression of respiration by a high concentration of glucose) is apparent, and cells offered glucose now meet less
of their ATP demand through oxidative phosphorylation (Fig. 16.4B). This
326
effect is entirely absent, however, upon normalizing to the basal rate

(Fig. 16.4E). Finally, a small amount of the complex III inhibitor
myxothiazol slightly decreases the basal rate of respiration and reduces maximal respiration in primary rat cortical neurons (Fig. 16.4C). When scaled to
the basal rate, the reduction in maximal respiration is minimized and the
small but measurable change in basal respiration is lost (Fig. 16.4F).
2.3. Using permeabilized cells to localize bioenergetic changes

observed with intact cells
The analysis described in Section 2.1 is a powerful tool for diagnosing global
changes in cellular energy metabolism. The assay can reveal altered rates of
cellular ATP utilization, efficiency of oxidative phosphorylation, and maximal capacity for substrate oxidation. Pinpointing the precise pathway or
mechanism responsible for such changes, however, is often restricted when
studying respiration in intact cells. Many cell types possess internal stores of
glycogen, triglyceride, and other endogenous substrate pools, so the substrates provided in the assay medium often do not exclusively fuel respiration. Moreover, several respiratory substrates, nucleotides, and specific
inhibitors that could be used to identify mechanisms of action are poorly cell
permeant. In specific cases, membrane-permeant substrate analogs can be
used to circumvent this problem (Weinberg et al., 2010), though their
breakdown into the active metabolite can often limit the rate of respiration
(Clerc & Polster, 2012; Divakaruni et al., 2013).
Plasma membrane permeabilization (forming large pores in the cellular
plasma membrane to deliver soluble molecules to in situ organelles) can
overcome these challenges. Substrates, inhibitors, and co-factors can be
offered directly to mitochondria for pathway-specific analysis. Since the
plasma membrane has a disproportionately high cholesterol content
(Fiskum, Craig, Decker, & Lehninger, 1980), amphipathic glycosides that
bind to cholesterol such as digitonin and saponin can be used to permeabilize
the plasma membrane with some selectivity. If used, these must be precisely
titrated as they can also form pores in the mitochondrial outer membrane
and allow cytochrome c release. However, use of a recombinant, mutant
perfringolysin O [rPFO; commercially XF Plasma Membrane Permeabilizer
(XF PMP; Seahorse Bioscience)] is far less prone to outer membrane
permeabilization or cell lifting than detergent-based approaches. This
cholesterol-dependent cytolysin has been used to reveal mitochondrial
mechanisms of drug action (Divakaruni et al., 2013) and distinguish between
effects on substrate transport at the plasma membrane or mitochondrial inner
327
membrane (Diers et al., 2014). Thorough protocols and trouble-shooting

recommendations for setting up these experiments are available elsewhere
(Divakaruni et al., 2014).
Figure 16.5 highlights the power of substrate-specific analysis in permeabilized cells to identify the mechanism by which a drug or genetic modification may affect cellular metabolism. Altered activity of multiple regulatory
points in cellular metabolism can yield analogous bioenergetic profiles in
intact cells. For example, inhibition of the electron transport chain (rotenone
as an example, Fig. 16.5B), substrate transport (UK5099; Fig. 16.5D), or ratecontrolling enzymes upstream of the TCA cycle (BPTES; Fig. 16.5F) can all
reduce ATP-linked and maximal respiration in whole cells. Because respiratory substrates feed into the electron transport chain via different pathways,
cells can be permeabilized and offered specific substrates to pinpoint the
enzyme or pathway responsible (see Fig. 16.5A as an overview).
For example, respiratory inhibition in cortical neurons by rotenone
(an inhibitor of the quinone-binding site of complex I) can be localized
to dysfunctional NADH-linked respiration with the careful selection of
substrates. Figure 16.5C shows oxidation of three different NADH-linked
substrates collapses but succinate oxidation (which bypasses complex I) is
unchanged, rendering complex I dysfunction as the most likely mechanism
of action of the inhibitor. Defects in substrate-specific transport can also be
identified. Addition of UK5099, an inhibitor of pyruvate uptake into the
matrix by the mitochondrial pyruvate carrier (MPC; Halestrap, 1975),
can generate a profile almost identical to submaximal complex
I inhibition by rotenone in cortical neurons (Fig. 16.5B and D). Permeabilized cell respirometry, however, shows that oxygen consumption is
unaffected when supported by glutamate or methyl pyruvate, a pyruvate
analog that crosses the mitochondrial inner membrane in a permeabilized
neuron (Fig. 16.5E). The result pinpoints the effect to facilitated pyruvate
uptake into the matrix by the MPC.
Other enzymes distinct from the electron transport chain, TCA cycle, or
substrate transport can also control the rate of intact cell respiration under
appropriate conditions. For example, the glutaminase inhibitor BPTES
(Robinson et al., 2007) can restrict the rate of both ATP-linked and maximal
respiration in whole A549 lung carcinoma cells (Fig. 16.5F). However,
BPTES does not affect TCA cycle flux or activity of the electron transport
chain, as oxidation of pyruvate, glutamate, and succinate is unchanged. It is
only when permeabilized cells are offered glutamine (with malate) that a
defect is observed (Fig. 16.5G). Although the inhibition is slight, this is
Figure 16.5 Using permeabilized cells to follow up results observed in intact cells and pinpoint mechanisms of action. (A) Schematic of the
respiratory chain showing sites of inhibition by rotenone (respiratory complex I), UK5099 (mitochondrial pyruvate carrier), and BPTES (glutaminase). Respiratory substrates used in C, E, and G are presented in bold and boxed. All data in (B)-(G) are from a single biological replicate
and presented as mean S.E.M. from a minimum of five wells (B) Primary cortical neurons were maintained and assayed as in Fig. 16.2D with
the following exceptions: cells were used 12 days after plating and, where indicated, offered 10 nM rotenone for 1 h prior to taking initial
measurements. Arrows (in order) indicate addition of 2 g/mL (final) oligomycin, 400 nM (final) FCCP, and 1 M (final) rotenone with 2 M
(final) antimycin A. (C) Primary neurons were permeabilized with 3 nM XF Plasma Membrane Permeabilizer (XF PMP) in MAS buffer supplemented with 0.2% BSA (Divakaruni et al., 2014) and, where indicated, treated with 1 M rotenone immediately prior to the measurements.
Maximal respiration was taken as the difference between uncoupler-stimulated respiration (2 M FCCP final) and respiration in the presence
of rotenone and antimycin A (1 and 2 M, respectively). Pyr/Mal: 5 mM pyruvate, 1 mM malate; Glu/Mal: 10 mM glutamate, 10 mM malate;
-HB/Mal: 10 mM -hydroxybutyrate, 1 mM malate; Succ/Rot: 10 mM succinate, 2 M rotenone. (D) Neurons were treated analogously to
(B) except, where indicated, 2 M UK5099 was added for 1 h prior to taking initial measurements. (E) Neurons were permeabilized and
assayed as in (C). MePyr/Mal: 20 mM methyl pyruvate, 5 mM pyruvate, 1 mM malate. (F) A549 cells were maintained as in Fig. 16.2C. Cells
were plated at 1 104 cells/well, grown for 2 days, and respiration was measured in medium containing 8 mM glucose and 4 mM glutamine.
Where indicated, 3 M BPTES was added 30 min prior to taking initial measurements. Arrows are as in (B). [FCCP] 400 nM. (G) A549 cells were
permeabilized as in (B). Gln/Mal: 10 mM glutamine, 10 mM malate. Mal: 10 mM malate.
330
probably due to the relatively unique capacity of these cells to sustain high
rates of respiration on malate alone. Surprisingly, the respiration rate on glutamine was greater than on glutamate, which likely indicates that substrate
transport (rather than oxidation) controlled the rate of glutamate-driven respiration in this cell line.
Even if a substrate-specific effect is not observed, consistent changes in
respiration driven by a range of substrates may also be informative. Increased
rates on all substrates may be attributable to changes in mitochondrial biogenesis or ultrastructure (e.g., increased cristae density). Decreased respiration on all substrates may be similarly indicative of derangements in
mitochondrial biogenesis, dynamics, or ultrastructure. Artificially delivering
electrons to cytochrome oxidase via TMPD (added with ascorbate to ensure
its constant reduction) can indicate whether reductions in the rate are also
simply due to complex IV dysfunction. Work with isolated mitochondria
suggests complex IV is typically not a limiting factor of the respiratory rate
under most circumstances (Davey, Peuchen, & Clark, 1998), so increases in
the rate of respiration on all substrates are likely unattributable to increased
cytochrome oxidase activity given our current understanding of respiratory
chain function. Measurements of cytochrome oxidase activity in the XF
Analyzer are generally qualitative and should be followed by specific inhibition of the enzyme with azide.
Observing no difference in substrate oxidation in permeabilized cells
can also be instructive when a drug or genetic modification elicits a change
in the bioenergetic profile of intact cells. A lack of an effect can indicate a
mechanism extrinsic to mitochondrial function, and suggests several possibilities including changes in substrate transport across the plasma membrane
(Diers et al., 2014), glycolytic provision of pyruvate to mitochondria, breakdown of endogenously stored substrates, cytoplasmic signaling events that
affect respiration, or the requirement to metabolize a drug into its
active form.
3. THE EXTRACELLULAR ACIDIFICATION RATE

Plate-based technologies are increasingly used to measure proton
efflux alongside respiration and have helped foster a wider appreciation
for the role of aerobic glycolysis in physiology and disease (Chang et al.,
2013; Frezza et al., 2011; Teperino et al., 2012). Although literature regarding oxygen consumption measurements to study mitochondrial function is
abundant, there is a scarcity of information about measuring glucose
331
oxidation by the rate of medium acidification in plate-based assays. As such,

Section 2 offers practical guidelines and additional perspective to an existing
body of work (notably Brand & Nicholls, 2011; Hill et al., 2012). Section 3,
however, focuses on the biochemistry and sources of acid production
from glucose oxidation. We believe the dearth of readily accessible information regarding measurements of the extracellular acidification rate (ECAR)
requires first establishing the foundations for the measurements. Initially, we
provide the core principles for why glycolytic turnover correlates with cellular proton efflux, and later address the contribution of respiratory CO2
evolution to medium acidification. Finally, we place this analysis within
the context of cellular ATP demand to show that rate-based measurements
of respiration and extracellular pH can broadly characterize an energetic
phenotype.
When measuring glucose oxidation in a plate-based assay, both glycolysis
and respiration will acidify the assay medium. At the most basic level, without considering individual reactions and leaving aside anabolism and biosynthesis, cellular ATP production by glucose uptake and oxidation produces
acidic metabolic end products. During glycolysis, the breakdown of
one uncharged glucose molecule into two pyruvate anions (pKa 2.5)
requires the release of two H+ at physiological pH. The ultimate fate of
the carbon is the efflux of two lactate anions (pKa 3.9) along with two
H+ to the experimental medium (the precise source of proton production
is discussed in Section 3.1). When glucose-derived pyruvate is linked to oxidative phosphorylation, CO2 is evolved, hydrated to carbonic acid in an
aqueous medium, and released as bicarbonate (HCO3 ) with H+
(Section 3.2).
As such, the catabolism of a six-carbon glucose molecule into two lactate
anions releases two protons to the medium, while oxidation of glucosederived pyruvate by matrix dehydrogenases releases six protons to the
medium for each glucose molecule. It is essential to view these relative contributions of proton production in the context of a steady-state ATP
demand. Two ATP molecules are produced and hydrolyzed when a glucose
molecule is converted to two lactate anions, whereas the maximum yield for
glucose carbon routed through oxidative phosphorylation is 33.5 ATP molecules (see Brand, 2005 with adjusted values from Watt, Montgomery,
Runswick, Leslie, & Walker, 2010). Therefore, meeting a steady-state
ATP demand exclusively by glycolysis (1 H+/ATP) is considerably more
acidifying than meeting the same demand strictly by oxidative phosphorylation (0.18 H+/ATP).
332
3.1. Medium acidification associated with glycolytic turnover

It is well established that glycolytic turnover is associated with acidification
of the extracellular medium, but precisely why this occurs is largely overlooked. This topic has been considered several times in the wider physiological contexts of exercising muscle (B
oning, Strobel, Beneke, & Maassen,
2005; Kemp, 2005; Robergs, Ghiasvanad, & Parker, 2004; Robergs &
Parker, 2005), general metabolism (Alberti & Cuthbert, 1982), the ischemic
heart (Dennis, Gevers, & Opie, 1991; Gevers, 1977), and energy metabolism
of lower organisms (Busa & Nuccitelli, 1984; Hochachka & Mommsen,
1983). The basic principles from this work still hold when discussing metabolism at the level of individual cell populations and are addressed in this
section.
The canonical EmbdenMeyerhofParnas pathway of glycolysis is a
series of 10 reactions that breaks down one molecule of glucose into two
molecules of pyruvate. Without considering the individual reactions themselves, it can be inferred from the structural changes alone that the catabolism
of uncharged glucose (C6H12O6) into two pyruvate anions (C3 H3 O3 1 )
is acidifying. Pyruvate exists overwhelmingly as the anion at physiological
pH (Fig.16.6). LDH converts pyruvate to lactate, regenerating NAD+
and allowing glycolytic flux to disengage from mitochondrial oxidation
of the pyridine nucleotide (NAD+/NADH) pool. As such, a byproduct
of increased glycolytic turnover is often a concomitant rise in lactate production. It is essential to note that the conversion of pyruvate to lactate is not
directly responsible for proton production associated with glycolysis.
Lactate (pKa 3.9) is the conjugate base of a weaker acid than pyruvate, meaning the LDH reaction is alkalinizing and helps buffer the protons produced
during glucose catabolism. Moreover, the pKas of pyruvate and lactate indicate the glycolysis-derived protons are produced well before lactate
(Fig. 16.6).
3.1.1 Proton release from glycolytic turnover
Figure 16.6 lists the reactions involved in glycolysis. From a broad perspective, only the initial and end products need to be considered when accounting for glucose-derived proton production. The conversion of glucose into
lactate, irrespective of the reactions, necessitates the release of two protons.
A more full interpretation is gained from considering the steps involved and
can be helpful because:
Figure 16.6 Proton production from the catabolism of glucose to lactate. Protons released or consumed are listed in red (dark gray in print
version). The phosphoglycerate kinase reaction is emphasized for two reasons: (i) it is the first reaction in which a carboxylic acid salt is made,
and (ii) it produces ATP without concomitant proton production. Values for pKas of three-carbon metabolites are taken from Robergs et al.
(2004).
334
(i) Particularly in proliferating cells, glucose-derived carbon is often diverted from glycolytic reactions to drive several biosynthetic pathways
without concomitant lactate production (e.g., glucose-6-phosphate
used for nucleotide biosynthesis or 3-phosphoglycerate for serine production; Lunt & Vander Heiden, 2011).
(ii) In plate-based assays, cells are often assayed in a complex medium with
multiple oxidizable substrates that feed into oxidative pathways either
up- or downstream of proton-producing reactions (e.g., offering cells
both glucose and pyruvate).
Overall, six hydrogens are liberated from the glucose backbone during its
catabolism into pyruvate, and the reactions ultimately result in two water
molecules and two free protons released to the medium. The specific reactions involved in the accounting of hydrogens are as follows:
HexokinaseA proton is released from the hydroxyl group on the sixth
carbon of glucose (C6) as glucose is converted to glucose-6-phosphate.
Hydrogens released from glucose carbon backbone: 1 as H+.
Phosphofructokinase (PFK)An analogous reaction occurs at C1 of
fructose-6-phosphate to release another proton to the medium.
Hydrogens released from carbon backbone: 1 as H+.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)A crucial regulatory
point in glycolysis, the GAPDH reaction converts glyceraldehyde-3phosphate (G-3-P) into the acyl phosphate 1,3-bisphosphoglycerate
(1,3-BPG). In the first step of the reaction, a hydride ion (H) from C1
of G-3-P is used to reduce NAD+. The subsequent phosphorylation of
C1 to form 1,3-BPG involves the release of a proton from inorganic phosphate (Pi) to form the phosphoester linkage.
Hydrogens released from carbon backbone: 2 as H (1 for each G-3-P). Two H+
are also released from Pi.
EnolaseDuring the enolase reaction, two hydrogens are lost from
2-phosphoglycerate (2-PG) as a water molecule is released during the
formation of phosphoenolpyruvate.
Hydrogens released from carbon backbone: 4 as H2O (2 for each 2-PG).
Pyruvate kinaseAfter conversion of phosphoenolpyruvate to pyruvate
with concomitant production of ATP, the rapid tautomerization of
pyruvate from its enol to its keto form consumes a proton.
Hydrogens added to carbon backbone: 2 as H+ (1 for each pyruvate).
As it relates to hydrogen, the net effect of glycolysis on the carbon backbone
of glucose is the loss of four hydrogen species released as water molecules and
two hydride ions that reduce two NAD+ molecules. Two protons are
335
released from the carbon skeleton during the hexokinase and PFK reactions,
but two are also added back during keto-enol tautomerization. Accounting
for the protons lost from inorganic phosphate during the GAPDH reaction,
the net catabolism of glucose into pyruvate releases two protons to the
cytoplasm.
It is important to note that, although the hexokinase and PFK reactions
produce protons via ATP hydrolyzing reactions, the production of
ATP during the phosphoglycerate kinase reaction does not consume a
proton and does not alter the cytoplasmic pH. The same is true for the
phosphorylation reaction of pyruvate kinase, although the subsequent,
nonenzymatic tautomerization of pyruvate consumes two protons. The
phosphoglycerate kinase reaction is also the first reaction in which a carboxylic acid salt, 3-phosphoglycerate, is produced. Because the carboxyl
moiety is formed during substrate-level phosphorylation, a proton is not
lost to the medium from either this or any of the subsequently formed carboxylates. Each carboxylic acid species produced during glycolysis exists as
the acid salt, and the three acidifying reactions occur prior to the production of 3-phosphoglycerate.
Pyruvate sits at the branch point between oxidative phosphorylation and
the LDH reaction that allows increased glycolytic flux. Upon production,
pyruvate can have several metabolic fates, though the predominant ones
are facilitated transport into mitochondria or conversion to lactate or alanine
in the cytoplasm. LDH couples the reduction of pyruvate to lactate with the
oxidation of NADH to NAD+, the latter being necessary for GAPDH activity (Fig. 16.6). NAD+ is also regenerated by activity of the malate-aspartate
and glycerol-3-phosphate shuttles, which link the reducing equivalents of
glycolytically derived NADH with the mitochondrial electron
transport chain.
3.1.2 ATP hydrolysis
As discussed in Section 3.1.1, the conversion of pyruvate to lactate is alkalinizing and not the source of proton production during glycolysis. As shown
in Fig. 16.6, the reactions converting a glucose molecule into two lactate
molecules produce four protons (hexokinase, PFK, and GAPDH reactions)
and consume four protons (pyruvate kinase and LDH). What, then, is the
major source of medium acidification during periods of high glycolytic turnover that yields the well-known reaction of glucose ! 2 lactate + 2H+? We
support the view of several others (Alberti & Cuthbert, 1982; Busa &
Nuccitelli, 1984; Gevers, 1977; Hochachka & Mommsen, 1983; Robergs
336
et al., 2004): the fully balanced reaction for steady-state glycolytic flux
requires considering the hydrolysis of the two ATP molecules produced
during oxidation of glucose into lactate (Fig. 16.6). ATP hydrolysis is acidifying, and is used to drive dozens of processes such as macromolecule biosynthesis, active transport across membranes and ion homeostasis, and
maintenance of cell junctions and shape.
At neutral pH, for every 2 mol of ATP hydrolyzed, one proton is
released to the medium (inorganic phosphate can buffer a proton; Eqs. (16.1)
and (16.2); Hochachka & Mommsen, 1983; Robergs et al., 2004).
2MgATP2 + 2H2 O ! 2MgADP + 2HPO4 2 + 2H + pH > 8:0
(16.1)
2
2 MgATP
+ 2 H2 O ! 2 MgADP + HPO4
2
+ H2 PO4 + H
pH 7:0
(16.2)
An elegant proof of principle using the XF Analyzer is presented in

McQuaker, Quinlan, Caldwell, Brand, and Hartley, (2013). The FoF1
ATP synthase is an entirely reversible enzyme, though in most experimental
designs it runs in the direction of ADP phosphorylation because of the constant generation of a mitochondrial membrane potential. When the potential drops below a threshold level, however, the enzyme can act as an ATPase
(Nicholls & Ferguson, 2013). It hydrolyzes ATP to generate a membrane
potential, much like how some bacteria hydrolyze ATP to generate a proton
gradient that drives active solute transport or flagellar motion.
In this experiment, isolated mitochondria were offered antimycin
A (blocking electron transfer) along with excess ATP. Upon addition of
FCCP, the medium sharply acidified, owing to the hydrolysis of ATP by
the ATP synthase to generate a membrane potential. The interpretation is
reinforced by addition of oligomycin, inhibiting activity of the enzyme
and causing the medium pH to return to the initial value. Indeed, the converse can also be shown, as isolated mitochondria phosphorylating ADP
(i.e., state 3 respiration) alkalinize the experimental medium (Rogers
et al., 2011; Vaghy, 1979).
The balanced reactions presented in Fig. 16.6 demonstrate that it is
essential to account for the relationship between the production of ATP
and its consumption. Assuming a 1:1 coupling between ATP production
and consumption (i.e., the steady-state ATP:ADP ratio is constant), the
net production of H+ during glycolytic turnover is clear. Although the figure presented is an oversimplification, the complicating factors (free [Mg2+],
337
[Pi 2 ], and physiological pH) change the stoichiometry of the individual

H+-producing steps but not the overall result: catabolism of one molecule
of glucose by glycolysis produces two protons (Busa & Nuccitelli, 1984).
The accounting of proton production from glucose oxidation changes if,
instead of producing lactate, glucose-derived pyruvate is fully oxidized to
CO2. When glucose-derived pyruvate is coupled to oxidative phosphorylation, there is normally a tight coupling between H+ production and consumption in vivo. Intracellular pH is kept within the range of homeostatic
buffering capacities and is therefore essentially constant (Alberti &
Cuthbert, 1982; Hochachka & Mommsen, 1983; McConnell et al., 1992;
Zilva, 1978). Upon entry into mitochondria, the carbon backbone of pyruvate is released as CO2 in the matrix by pyruvate dehydrogenase (PDH) and
TCA cycle enzymes. CO2 is a volatile acid, buffered in vivo by the bicarbonate buffering system, and eliminated by exhalation via the lungs.
The intracellular proton concentration is balanced by maintenance of the
steady-state ATP:ADP ratio and redox poise of cytochromes and co-factors
during oxidative phosphorylation (Hochachka & Mommsen, 1983). As a
cursory example, each of the products of cytoplasmic ATP hydrolysis
(ADP, Pi, H+) is transported into the matrix for re-synthesis of ATP.
A simplified, balanced equation for oxidative catabolism of glucose into
six molecules of CO2 (glycolysis, PDH, TCA cycle, and electron transport
chain) coupled to ADP phosphorylation is given in Eq. (16.3). Considering
cellular ATP demand and maintenance of a steady-state ATP:ADP ratio
(Eq. 16.4) yields the balanced reaction for oxidative phosphorylation
(Eq. 16.5; CO2 is assumed to be eliminated in vivo). A detailed accounting
of the reactions is presented in Robergs and Parker (2005), and the effects of
pH on stoichiometry are addressed in Busa and Nuccitelli (1984).
C6 H12 O6 + 6 O2 + 33:5 MgADP + 33:5 HPO4 2 + 33:5 H +
! 6 CO2 + 33:5 MgATP2 + 39:5 H2 O
(16.3)
33:5 MgATP2 + 33:5 H2 O ! 33:5 MgADP + 33:5 HPO4 2 + 33:5 H +
C6 H12 O6 + 6 O2 ! 6 CO2 + 6 H2 O
(16.4)
(16.5)
The tight coupling between proton production and consumption breaks

down when glycolytic production of pyruvate is disengaged from oxidative
phosphorylation. The balance between ATP hydrolysis and synthesis again
provides an illustrative example. In glycolysis, protons are not stoichiometrically consumed during regeneration of ATP by phosphoglycerate kinase
338
and pyruvate kinase (see Fig. 16.6), leading to a drop in intracellular pH during glycolytic turnover.
Pancreatic -cells are a convenient model system to study the effects of
medium acidification when pyruvate is disproportionately funneled into
mitochondrial metabolism. They are characterized by low expression and
activity of LDH, and consequently channel glucose-derived pyruvate to
the mitochondria to precisely coordinate glucose-stimulated insulin secretion (Ainscow, Zhao, & Rutter, 2000; Sekine et al., 1994). Medium
acidification in both MIN-6 cells and two INS-1 clones is quite low
(El-Azzouny, Evans, Treutelaar, Kennedy, & Burant, 2014; Nishiki
et al., 2013; Sakamaki et al., 2014). As a broad, internally normalized
method of comparison, the OCR:ECAR ratio in these cells when offered
glucose (1722 mM) was over 30 for each of these cell types. As a point of
comparison, the ratios in the A549 cells and primary neurons discussed in
Fig. 16.8 were roughly two (A549s in unbuffered DMEM) and eight
(primary neurons in aCSF medium supplemented with 5 mM HEPES).
Data suggest the low but measurable rates of acidification seen in -cells
are attributable to CO2 evolution in a cell monolayer (discussed further
in Section 3.2).
3.1.3 Extracellular pH as a measure of glycolytic turnover using
Seahorse XF technology
The efflux of protons by the cell to maintain intracellular pH can be used as
an indirect measurement of intracellular proton production. Put another
way, extracellular glucose is taken up by the cell and converted into
acidic end products (lactate or bicarbonate) that are ultimately effluxed
with protons, rendering medium pH as an indirect measurement of the relevant oxidative pathway. Indeed, measurement of both intra- and extracellular pH as an indicator of glycolysis has been conducted for decades with pH
microelectrodes (Balaban & Bader, 1984; Newell, Franchi, Pouyssegur, &
Tannock, 1993), a silicon-based potentiometric sensor (McConnell et al.,
1992), and 31P NMR (Allen, Morris, Orchard, & Pirolo, 1985). Several protein families including Na+/H+ exchangers (Mahnensmith & Aronson,
1985) and H+-linked monocarboxlyate transporters (Halestrap, 2013) regulate intracellular pH by proton efflux (Parks, Chiche, & Pouyssegur, 2013).
Although the lactate anion is not the source of extracellular acidification,
but rather an associated byproduct, medium acidification and lactate efflux
often qualitatively track under steady-state, basal conditions. The correlation
has been extensively reported in the literature, and has been observed well
339
before the development of plate-based methodologies. As an example, the

rate of medium acidification correlates with lactate production in suspended,
ras-transfected Chinese hamster lung fibroblasts, and the correlation holds
when the glycolytic rate is either increased by hypoxia or decreased by
defective glucose uptake and phosphoglucose isomerase activity (Newell
et al., 1993).
Using Seahorse XF technology, changes in basal ECAR in adherent cell
monolayers have been correlated to end-point measurements of lactate in
spent medium (reflecting total lactate efflux) in dozens of instances for
immortalized cells. A bulk of these comparisons have been made in models
of cancer (Blouin et al., 2010; Chan et al., 2011; Delgado et al., 2010; Frezza
et al., 2011; Liu et al., 2014; Mouradian et al., 2014; Xie et al., 2009). The
qualitative relationship between basal ECAR and snapshot-in-time lactate
levels has also been shown for myocytes (Teperino et al., 2012), adipocytes
(Keuper et al., 2014; Teperino et al., 2012), and immune cells (Everts et al.,
2014; Gubser et al., 2013; Macintyre et al., 2014).
Quantitative comparisons between ECAR and lactate levels, however,
should consider the following:
(i) The XF Analyzer measures a rate and end-point metabolite assays
report a fixed, accumulated total.
(ii) The instrument reports ECAR on a logarithmic scale (mpH/min), and
the medium buffering capacity must be accounted for to translate this
value into a total amount of protons. This can be easily done by entering a buffering capacity in the XF/Wave software or by calibration
with acid via the injector ports on the XF Analyzer (McQuaker
et al., 2013).
(iii) Glycolytic intermediates are required for the biosynthesis of nucleotides, lipids, and amino acids. Particularly in proliferating cells, acidifying reactions associated with glycolysis can occur without the
concomitant production of lactate (Lunt & Vander Heiden, 2011).
(iv) Respiratory CO2 evolution can contribute to the rate of medium acidification (Section 3.2).
As described above, the correlation between end-point measurements of
lactate efflux and basal rates of medium acidification has been independently
established for several cell types. To our knowledge, though, there has yet to
be a rigorous validation of ECAR in response to acutely added effectors on
the time scale of an XF assay. This demonstration would be important, as
one of the singular features of the XF Analyzer is the ability to acutely inject
effectors into the well and monitor the kinetics of how oxygen consumption
340
and medium pH change in response to substrates or pharmacologic agents.

This may prove to be technically challenging, as the sensitivity of most endpoint metabolite measurements may be insufficient to address changes in lactate efflux that occur over the course of minutes.
The limited work that is available, though, is encouraging. Acute
increases in ECAR in response to oligomycin correlate well with lactate
accumulated in the extracellular medium collected 3 h after treatment
(Keuper et al., 2014). Moreover, acutely changing medium composition
during measurements of extracellular acidification with a microphysiometer
demonstrate that large changes in glycolytic flux can change medium pH
in minutes under appropriate conditions. Suspended CHO-K1 cells give
a stable rate of extracellular acidification when 10 mM glucose is offered
as a substrate, and that rate drops to 25% of its initial rate when glucose
is replaced with pyruvate, 2-deoxyglucose, or no substrate (McConnell
et al., 1992). It should be noted that glycogenolysis can occur under all
three of the conditions tested and may account for some fraction of the
remaining rate.
Those who use the XF Analyzer with recently developed plates to study
bioenergetics of spheroids (http://www.seahorsebio.com/learning/webinars/
details.php?wID63) should be aware that the correlation between medium
acidification and lactate production may not be as applicable in multicellular
structures (Newell et al., 1993) and in vivo (Helmlinger, Sckell, Dellian,
Forbes, & Jain, 2002). Perhaps surprisingly, ras-transfected cells deficient
in glycolysis that exhibit reduced lactate production and medium acidity
in vitro still form acidic tumor microenvironments when injected into
mice. The acidity is present despite an appreciable drop in accumulated lactate, suggesting glycolytic turnover cannot entirely explain tumor acidity
(Newell et al., 1993). This conclusion has been reinforced in a rat glioblastoma model by a combination of magnetic resonance spectroscopy and
metabolite mapping (Garca-Martn et al., 2001). Rather than glycolytic
turnover, it is widely thought that CO2 production from oxidative mitochondrial metabolism is a large contributor to solid tumor acidity (Gillies,
Robey, & Gatenby, 2008; Newell et al., 1993). In vitro models reinforce
these conclusions. Spheroids formed with HCT116 colon carcinoma cells
transfected with carbonic anhydrase 9, an isoform associated with cancer
and upregulated under hypoxia, suggest CO2 is a primary contributor of
extracellular acidification in three-dimensional cancer models (Sweitach,
Patiar, Supuran, Harris, & Vaughan-Jones, 2009).
341
3.2. Medium acidification not attributable to glycolytic

turnover
3.2.1 CO2 production
The complete breakdown of one molecule of glucose through oxidative
phosphorylation results in the evolution of six molecules of carbon dioxide
(one each from PDH, isocitrate dehydrogenase, and -ketoglutarate dehydrogenase for every molecule of pyruvate). Formation of CO2 in an aqueous
environment causes its reversible hydration and dissociation into bicarbonate and a proton (Eq. 16.6).
H2 O + CO2 H2 CO3 H + + HCO3
(16.6)
This may be particularly relevant in primary cells and differentiated cell

culture models, as glycolytic turnover is generally not as robust as it is in
immortalized or proliferative cells.
In synaptoneurosomes, both glycolytic turnover and CO2 evolution
appear to contribute to ECAR (Choi et al., 2009). When oxidative phosphorylation is blocked by addition of oligomycin, medium acidification
increases when glucose is provided as a substrate but not pyruvate,
reflecting increases in glycolytic turnover to meet energetic needs (ATP
production and pyridine nucleotide turnover). However, injection of
FCCP causes ECAR to increase when either glucose or pyruvate is offered
as the respiratory substrate. This increase tracks well with the respiratory
rate and is likely due to CO2 evolution from increased PDH and TCA
cycle flux (addition of electron transport chain inhibitors to halt TCA cycle
flux can reinforce such an interpretation, addressed in Section 3.2.2
and Fig. 16.7).
Work published by Sakamaki and colleagues (Sakamaki et al., 2014)
using the glucose-responsive MIN6 cells provides perhaps the most convincing demonstration that CO2 evolution can contribute to the ECAR signal. An acidification rate is observed and acutely increases about fourfold
when the glucose concentration is increased from 1 mM to 20 mM.
Although this may appear to reflect glycolytic turnover, the rate of medium
acidification immediately collapses to zero upon injection of oligomycin
(Fig. S4g; Sakamaki et al., 2014). Given the minimal LDH expression in
pancreatic -cells, the result is good evidence that CO2 evolution from
glucose-derived pyruvate is entirely responsible for the reported ECAR
in this system.
342
Figure 16.7 Qualitative assessment of the contribution of CO2 to ECAR. All cells were
maintained, plated, and assayed as described in Fig. 16.2. Assay medium was
unbuffered DMEM supplemented with 8 mM glucose, 3 mM glutamine, and 3 mM pyruvate. Concentrations of oligomycin, rotenone, and antimycin A are given in Fig. 16.2.
[FCCP] 600 nM for A549, 1 M for C2C12 myoblasts, and 1.2 M for C2C12 myotubes.
All data are from a single biological replicate and presented as mean S.E.M. from a
minimum of five wells.
Because the contribution of CO2 to ECAR is likely dependent on several factors, including perhaps carbonic anhydrase expression, cellular substrate preference, and medium composition, it is almost assuredly different
across cell types. Moreover, experimental perturbations during the course of
a given assay are likely to change the degree to which it contributes to the
rate. For example, if ATP demand is high, oligomycin will cause glycolysis
to increase but will slow TCA cycle flux. Cellular substrate preference and
rates of anabolism may also affect the contribution of CO2 to extracellular
acidification. The most straightforward example would be the differential
amounts of CO2 evolved for each molecule of oxygen consumed during
purely fat (0.7), glucose (1.0), or pyruvate (1.2) oxidation.
3.2.2 Qualitative assessment of the CO2 contribution to ECAR
Observing the ECAR recordings during the standardized oxygen consumption measurements described in Section 2.1 may provide a cursory, empirical estimate of whether mitochondrially derived CO2 is a confounding
variable to interpreting ECAR as glycolytic flux (Fig. 16.7). After
oligomycin injection (when TCA cycle flux is low in well coupled cells),
343
FCCP addition causes respiration to increase (with concomitantly high

TCA cycle flux) and subsequent inhibition of the respiratory chain with
rotenone and antimycin A will shut down electron transfer (virtually no
TCA cycle flux). In cells given oligomycin and FCCP, changes in ECAR
before and after injection of electron transport chain inhibitors may indicate
whether CO2 is a contributing factor in a given system. If ECAR declines in
response to rotenone and antimycin A, then TCA cycle activity was likely
contributing to the ECAR signal. If ECAR remains constant or increases
with addition of the respiratory inhibitors, then the contribution to the
ECAR signal may be primarily glycolysis.
Figure 16.7 shows three of the cell types used in Fig. 16.2 to qualitatively
assess the contribution of respiratory CO2 to ECAR (all cells were offered
glucose, pyruvate, and glutamine in the assay medium). Proliferating A549
cells (Fig. 16.7A) exhibit a robust ECAR signal when oligomycin is added.
The signal does not change much upon addition of FCCP, and transiently
increases when electron transport chain inhibitors shut down TCA cycle
flux. As there does not appear to be any correlation between ECAR and
CO2 evolution (indirectly gauged by oxygen consumption, see lower
panels), bicarbonate-associated acid production is probably only a small fraction of the signal in these highly glycolytic, proliferative cells. There appears
to be a modest contribution in C2C12 myocytes (Fig. 16.7B) under these
conditions, as ECAR drops upon addition of rotenone and antimycin A
but remains higher than the initial, basal rate. Post-mitotic, terminally differentiated C2C12 myotubes provide an example where a low basal ECAR
signal is likely mostly attributable to CO2 evolution. The rate of medium
acidification increases more than twofold upon addition of FCCP and collapses to below the basal rate when mitochondrial electron transfer is
inhibited. The result suggests that in primary or terminally differentiated
cells, where glycolysis is usually not high, CO2 evolution from matrix dehydrogenases can be a significant contributor to the rate.
This suggestion is not meant to be a quantitative test, and a relatively stable ECAR signal after protonophore addition is necessary for proper interpretation. Multiple complicating factors can confound the result such as
cytoplasmic acidification from FCCP slowing the rate of glycolysis
(Ereci
nska, Deas, & Silver, 1995; Parks, Chiche, et al., 2013; Parks,
Mazure, Counillon, & Pouyssegur, 2013), the incomplete shutdown of
TCA cycle flux by oligomycin, and perhaps a compromised ability of some
cells to transport and oxidize glucose when mitochondrial ATP production
is blocked. Some cells may not be able to maintain the plasma membrane
344
sodium gradient for glucose transport or sufficiently provide ATP for the
early reactions of glycolysis.
Although the use of inhibitors like 2-deoxyglucose, koningic acid,
iodoacetate, 3-bromopyruvate, and oxamate are often used to localize
ECAR to glycolytic turnover, any compound that slows the rate of glucose
catabolism into pyruvate can also limit the rate at which glucose-derived
pyruvate enters the TCA cycle. As such, if a component of ECAR is attributable to CO2 evolution from pyruvate oxidation, then changes in the rate
upon inhibitor addition are not strictly attributable to turnover of glycolytic
enzymes. Those seeking to use inhibitors of glycolysis should simply be
aware of this caveat, and experiments can be designed where pyruvate is
injected alongside glycolytic inhibitors to discriminate between effects on
glycolysis and pyruvate-derived CO2.
2-Deoxyglucose is the most commonly used glycolytic inhibitor in
plate-based experiments, and in some contexts its use can be difficult to
interpret. As a competitive inhibitor of hexokinase, is it often added at high
concentrations such as 100 mM that are known to affect cell osmolarity
within minutes (see Epstein, Xu, Gillies, & Gatenby, 2014). Effects of
slowing glycolytic flux can be difficult to separate from putative effects of
an osmotic crisis. Moreover, the breakdown of glycogen into pyruvate
bypasses hexokinase, so medium acidification from glycogenolysis is not
accounted for when using 2-deoxyglucose as an inhibitor. Rather than addition of supraphysiological concentrations of 2-deoxyglucose, we propose
that a combination of inhibitors at low concentrations may yield a more
interpretable result in these instances. Koningic acid is reportedly effective
in isolation (Hill et al., 2012), but can be prohibitively expensive.
3.2.2 Other contributing factors to ECAR
Work with a microphysiometer shows that transient changes in proton
efflux pathways, receptor activation state, and disequilibrium of monocarboxylic acid transport across the plasma membrane can have temporary
effects on the rate of medium extracellular acidification. The demonstration
reinforces the essential importance of interpreting ECAR data only when
the system has come to a new steady-state (i.e., a stable rate). Perhaps surprisingly, though, chronic knockdown of monocarboxylate transporter isoform 1 in KBM7 cells did not significantly alter either end-point
measurements of lactate efflux or basal ECAR, suggesting other isoforms
can maintain the balance of monocarboxylate uptake and efflux (Birsoy
et al., 2013). It is also formally possible that intracellular H+ buffering
345
capacities could be different between cell populations after targeted genetic

modification, though this has not been demonstrated to change the qualitative correlation between basal ECAR and lactate efflux.
3.3. Characterizing cellular ATP demand with respiration and

extracellular acidification
The ability to estimate the poise between ATP production via oxidative
phosphorylation and glycolysis with plate-based measurements allows for
a broad approximation of the rate of cellular ATP production. Several
assumptions must be made, including selection of a P/O ratio for ATPlinked respiration (the amount of ADP phosphorylated for each molecule
of O reduced; Brand, 2005), the contribution of CO2 evolution to ECAR,
and how the biosynthetic requirements of proliferation can separate activity
of glycolytic enzymes from ATP production (e.g., glucose-6-phosphate
used for nucleotide biosynthesis or 3-phosphoglycerate for amino acid biosynthesis). Nonetheless, these measurements can diagnose large shifts in the
balance between aerobic and anaerobic pathways to meet basal energy
demands (Birket et al., 2011; Keuper et al., 2014). When using the XF Analyzer, the researcher should take care to adjust ECAR values (reported in
mpH/min) into H+ produced per minute [Proton Production Rate], having
established the buffering capacity of the medium as discussed in
Section 3.1.3. OCR values should be adjusted to pmol O/min when calculating a P/O ratio (the instrument reports pmol O2/min).
Side-by-side analysis of OCR and ECAR can also be useful to phenotypically characterize cellular energetics. Figure 16.2 shows that proliferating
A549 lung epithelial cells make ATP at a rate much closer to their maximal
capacity than terminally differentiated cells such as cortical neurons.
Experiments can be designed with ECAR to similarly characterize cellular
energy demand. Oligomycin can stimulate an increase in glycolysis to meet
an endogenous ATP demand, but it may have a minimal effect in two
instances: (i) glycolysis is working closely to its maximal capacity in the basal
state or (ii) the basal glycolytic rate is limited by a low resting demand for
ATP. To distinguish between these two, an artificial ATP demand can
be imposed.
Figure 16.8 shows A549 cells and cortical neurons offered either
oligomycin (forcing glycolysis to meet the endogenous ATP demand) or
FCCP (imposing an artificially high ATP demand by converting mitochondria into ATP consumers; Section 3.1.2). After subsequent addition of rotenone and antimycin A to shut down TCA cycle flux, ECAR rates were
346
Figure 16.8 Determining and interpreting maximal extracellular acidification rates. Cortical neurons were maintained as described in Fig. 16.2D and extracellular acidification
was measured 14 days after plating in aCSF medium containing only 10 mM glucose.
Where indicated, either oligomycin (2 g/mL final) or FCCP (400 nM final) were acutely
added via the injector port. After two measurements, rotenone and antimycin
A (concentrations as in Fig. 16.2) were acutely added. Experiments with A549s were conducted similarly with cells grown at 1 104 cells/well for 2 days and assayed in
unbuffered DMEM supplemented with 10 mM glucose. Concentrations of oligomycin,
rotenone, and antimycin A were those used for neurons. [FCCP] 600 nM. Data are from
four biological replicates and presented as mean S.E.M. with a minimum of six technical replicates for each experiment.
recorded. The experiment reinforces the phenotypic characterization from

Fig. 16.2. Proliferative A549 cells with a high endogenous ATP demand
have a high ECAR relative to the oligomycin-stimulated rate (cells were
offered only glucose in this experiment, unlike in Fig. 16.7). Moreover,
addition of FCCP does not cause an appreciable increase in ECAR relative
to addition of oligomycin, indicating these A549 cells have a sufficiently
high ATP demand such that blocking mitochondrial ATP production
(+oligomycin) mimics an ATP crisis (+FCCP).
A different profile is obtained with cortical neurons. Oligomycin causes a
much larger proportional increase in ECAR over the basal rate than is seen
in A549 cells. Moreover, when an artificial ATP demand is induced, ECAR
proportionally increases far beyond the rate induced by oligomycin,
suggesting cortical neurons have a robust glycolytic capacity that is limited
by a low, resting ATP demand. A similar profile is observed in cortical synaptoneurosomes, where 5 M veratridine stimulates a larger proportional
increase in medium acidification than oligomycin (Choi et al., 2009).
The results from Figs. 16.2 and 16.8, along with established work regarding
347
the energetic changes associated with adipocyte maturation (Keuper et al.,

2014), provide a framework for measuring mitochondrial respiration and
medium acidification to characterize cellular energetics in a manner
corresponding with the differentiation state of cells.
SUMMARY
As use of plate-based technologies such as Seahorse XF Analyzers
increases, so too does the need to design and interpret experiments that maximize the information gained. Appropriately designed and controlled experiments with intact cells provide information about cellular ATP demand, the
efficiency of oxidative phosphorylation, the maximal capacity for mitochondrial substrate oxidation, and the relative balance between oxidative phosphorylation and glycolysis. When differences in the respiration rate are
observed, further experiments with permeabilized cells can diagnose the particular pathway(s) responsible for such a change. Moreover, an understanding of the sources of medium acidification during glycolysis and CO2
evolution can aid experimental interpretation as the appreciation of aerobic
glycolysis continues to grow.
ACKNOWLEDGMENTS
We gratefully acknowledge Drs. Martin Brand and Shona Mookerjee (Buck Institute for
Research on Aging), as well as Drs. Brian Dranka and George Rogers (Seahorse
Bioscience) for their helpful suggestions and careful reading of this chapter. We would
also like to thank Daniel Lamp (muscle fibers), Dr. Susanne Keipert (white adipocytes),
and Maria Kutschke (CLU201 astrocytes) for providing data. D. A. F. is Chief Scientific
Officer of Seahorse Bioscience. This work was supported by NIH R01NS087611 and
5P01 DK054441 (to A. N. M.), the German Center for Diabetes Research (DZD; to M. J.),
and Seahorse Bioscience.
REFERENCES
Affourtit, C., & Brand, M. D. (2009). Measuring mitochondrial bioenergetics in INS-1E
insulinoma cells. Methods in Enzymology, 457, 405424.
Affourtit, C., Jastroch, M. J., & Brand, M. D. (2011). Uncoupling protein-2 attenuates
glucose-stimulated insulin secretion in INS-1E insulinoma cells by lowering mitochondrial reactive oxygen species. Free Radicals in Biology and Medicine, 50, 609616.
Affourtit, C., Quinlan, C. L., & Brand, M. D. (2012). Measurement of proton leak and electron leak in isolated mitochondria. Methods in Molecular Biology, 810, 165182.
Ainscow, E. K., & Brand, M. D. (1999). Top-down control analysis of ATP turnover, glycolysis and oxidative phosphorylation. European Journal of Biochemistry, 263, 671685.
Ainscow, E. K., Mirshamsi, S., Tang, T., Ashford, M. L. J., & Rutter, G. A. (2002). Dynamic
imaging of free cytosolic ATP concentration during fuel sensing by rat hypothalamic
348
neurones: Evidence for ATP-independent control of ATP-sensitive K+ channels. Journal

of Physiology, 544, 429445.
Ainscow, E. K., Zhao, C., & Rutter, G. A. (2000). Acute overexpression of lactate
dehydrogenase-A perturbs -cell mitochondrial metabolism and insulin secretion.
Diabetes, 49, 11491155.
Alberti, K. G., & Cuthbert, C. (1982). The hydrogen ion in normal metabolism: A review.
In R. Porter, & G. Lawrenson (Eds.), Ciba Foundation Symposium 87: Metabolic Acidosis
(pp. 119). London, England: Pitman Books Ltd.
Allen, D. G., Morris, P. G., Orchard, C. H., & Pirolo, J. S. (1985). A nuclear magnetic resonance study of metabolism in the ferret heart during hypoxia and inhibition of glycolysis. Journal of Physiology, 361, 185204.
Balaban, R. S., & Bader, J. P. (1984). Studies on the relationship between glycolysis and
(Na++ K+)-ATPase in cultured cells. Biochimica et Biophysica Acta, 804, 419426.
Bedard, K., & Krause, K. H. (2007). The NOX family of ROS-generating NADPH oxidases: Physiology and pathophysiology. Physiological Reviews, 87, 245313.
Birket, M. J., Orr, A. L., Gerencser, A. A., Madden, D. T., Vitelli, C., Swistowski, A., et al.
(2011). A reduction in ATP demand and mitochondrial activity with neural differentiation of human embryonic stem cells. Journal of Cell Science, 124, 348358.
Birsoy, K., Wang, T., Possemato, R., Yilmaz, O. H., Koch, C. E., Chen, W. W., et al.
(2013). MCT-1 mediated transport of a toxic molecule is an effective strategy for
targeting glycolytic tumors. Nature Genetics, 45, 104108.
Blouin, M. J., Zhao, Y., Zakikhani, M., Algire, C., Piura, E., & Pollak, M. (2010). Loss of
function of PTEN alters the relationship between glucose concentration and cell proliferation, increases glycolysis, and sensitizes cells to 2-deoxyglucose. Cancer Letters, 289,
246253.
B
oning, D., Strobel, G., Beneke, R., & Maassen, N. (2005). Lactic acid still remains the real
cause of exercise-induced metabolic acidosis. American Journal of Physiology. Regulatory,
Integrative and Comparative Physiology, 289, R902R903.
Brand, M. D. (2005). The efficiency and plasticity of mitochondrial energy transduction. Biochemical Society Transactions, 33, 897904.
Brand, M. D., & Nicholls, D. G. (2011). Assessing mitochondrial dysfunction in cells. Biochemical Journal, 435, 297312.
Brand, M. D., Pakay, J. L., Ocloo, A., Kokoszka, J., Wallace, D. C., Brookes, P. S., et al.
(2005). The basal proton conductance of mitochondria depends on adenine nucleotide
translocase content. Biochemical Journal, 392, 353362.
Brookes, P. S., Rolfe, D. F. S., & Brand, M. D. (1997). The proton permeability of liposomes
made from mitochondrial inner membrane phospholipids: Comparison with isolated
mitochondria. Journal of Membrane Biology, 155, 167174.
Busa, W. B., & Nuccitelli, R. (1984). Metabolic regulation via intracellular pH. American
Journal of Physiology. Regulatory, Integrative and Comparative Physiology, 246, R409R438.
Buttgereit, F., & Brand, M. D. (1995). A hierarchy of ATP-consuming processes in mammalian cells. Biochemical Journal, 312, 163167.
Caro, P., Kishan, A. U., Norberg, E., Stanley, I. A., Chapuy, B., Ficarro, S. B., et al. (2012).
Metabolic signatures uncover distinct targets in molecular subsets of diffuse large B cell
lymphoma. Cancer Cell, 22, 547560.
Chacko, B. K., Kramer, P. A., Ravi, S., Johnson, M. S., Hardy, R. W., Ballinger, S. W., et al.
(2013). Methods for defining distinct bioenergetic profiles in platelets, lymphocytes,
monocytes, and neutrophils, and the oxidative burst from human blood. Laboratory Investigation, 93, 690700.
Chan, D. A., Sutphin, P. D., Nguyen, P., Turcotte, S., Lai, E. W., Banh, A., et al. (2011).
Targeting GLUT1 and the Warburg effect in renal cell carcinoma by chemical synthetic
lethality. Science Translational Medicine, 3, 94ra70.
349
Chang, C. H., Curtis, J. D., Maggi, L. B., Faubert, B., Villarino, A. V., OSullivan, D., et al.
(2013). Posttranscriptional control of T cell effector function by aerobic glycolysis. Cell,
153, 12391251.
Choi, S. W., Gerencser, A. A., & Nicholls, D. G. (2009). Bioenergetic analysis of isolated
cerebrocortical nerve terminals on a microgram scale: Spare respiratory capacity and stochastic mitochondrial failure. Journal of Neurochemistry, 109, 11791191.
Clerc, P., & Polster, B. M. (2012). Investigation of mitochondrial dysfunction by sequential
microplate-based respiration measurements from intact and permeabilized neurons.
PLoS One, 7, e34465.
Cunnane, S., Nugent, S., Roy, M., Courchesne-Loyer, A., Croteau, E., Tremblay, S., et al.
(2011). Brain fuel metabolism, aging, and Alzheimers disease. Nutrition, 27, 320.
Davey, G. P., Peuchen, S., & Clark, J. B. (1998). Energy thresholds in brain mitochondria.
Journal of Biological Chemistry, 273, 1275312757.
Deberardinis, R. J. (2014). Q&A: Targeting metabolism to diagnose and treat cancer. Cancer
Metabolism, 2, 5.
Delgado, T., Carroll, P. A., Punjabi, A. S., Margineantu, D., Hockenbery, D. M., &
Lagunoff, M. (2010). Induction of the Warburg effect by Kaposis sarcoma herpesvirus
is required for the maintenance of latently infected endothelial cells. Proceedings of the
National Academy of Sciences of United States of America, 107, 1069610701.
Dennis, S. C., Gevers, W., & Opie, L. H. (1991). Protons in ischemia: Where do they
come from; where do they go to? Journal of Molecular and Cellular Cardiology, 23,
10771086.
Diers, A. R., Broniowska, K. A., Chang, C. F., Hill, R. B., & Hogg, N. (2014).
S-Nitrosation of monocarboxylate transporter 1: Inhibition of pyruvate-fueled respiration and proliferation of breast cancer cells. Free Radicals in Medicine and Biology, 69,
229238.
Divakaruni, A. S., & Brand, M. D. (2011). The regulation and physiology of mitochondrial
proton leak. Physiology, 26, 192205.
Divakaruni, A. S., Rogers, G. W., & Murphy, A. N. (2014). Measuring mitochondrial function in permeabilized cells using the Seahorse XF Analyzer of a Clark-type oxygen electrode. Current Protocols in Toxicolocy, 60, 25.2.125.2.16.
Divakaruni, A. S., Wiley, S. E., Rogers, G. W., Andreyev, A. Y., Petrosyan, S.,
Loviscach, M., et al. (2013). Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier. Proceedings of the National Academy of Sciences of United States of
America, 110, 54225427.
Dmitriev, R. I., & Papkovsky, D. B. (2012). Optical probes and techniques for O2 measurement in live cells and tissue. Cellular and Molecular Life Sciences, 69, 20252039.
Dranka, B. P., Benavides, G. A., Diers, A. R., Giordano, S., Zelickson, B. R., Reily, C.,
et al. (2011). Assessing bioenergetic function in response to oxidative stress by metabolic
profiling. Free Radicals in Biology and Medicine, 51, 16211635.
Dranka, B. P., Hill, B. G., & Darley-Usmar, V. M. (2010). Mitochondrial reserve capacity in
endothelial cells: The impact of nitric oxide and reactive oxygen species. Free Radicals in
Biology and Medicine, 48, 905914.
Duchen, M. J. (1992). Ca2+-dependent changes in the mitochondrial energetics in single dissociated mouse sensory neurons. Biochemical Journal, 283, 4150.
El-Azzouny, M., Evans, C. R., Treutelaar, M. K., Kennedy, R. T., & Burant, C. F. (2014).
Increased glucose metabolism and glycerolipid formation by fatty acids and GPR40
receptor signaling underlies the fatty acid potentiation of insulin secretion. Journal of Biological Chemistry, 289, 1357513588.
Epstein, T., Xu, L., Gillies, R. J., & Gatenby, R. A. (2014). Separation of metabolic supply
and demand: Aerobic glycolysis as a normal physiological response to fluctuating energetic demands in the membrane. Cancer Metabolism, 2, 7.
350
Ereci
nska, M., Deas, J., & Silver, I. A. (1995). The effect of pH on glycolysis and phosphofructokinase activity in cultured cells and synaptosomes. Journal of Neurochemistry, 65,
27652772.
Everts, B., Amiel, E., Huang, S. C., Smith, A. M., Chang, C. H., Lam, W. Y., et al. (2014).
TLR-driven early glycolytic reprogramming via the kinases TBK1-IKK supports the
anabolic demands of dendritic cell activation. Nature Immunology, 15, 323332.
Fan, J., Kamphorst, J. J., Mathew, R., Chung, M. K., White, E., Shlomi, T., et al. (2013).
Glutamine-driven oxidative phosphorylation is a major ATP source in transformed
mammalian cells in both normoxia and hypoxia. Molecular Systems Biology, 9, 712.
Fillmore, N., & Lopaschuk, G. D. (2013). Targeting mitochondrial oxidative metabolism as
an approach to treat heart failure. Biochimica et Biophysica Acta, 1833, 857865.
Fiskum, G., Craig, S. W., Decker, G. L., & Lehninger, A. L. (1980). The cytoskeleton of
digitonin-treated rat hepatocytes. Proceedings of the National Academy of Sciences United
States of America, 77, 34303434.
Frey, T. G., Perkins, G. A., & Ellisman, M. H. (2006). Electron tomography of membranebound cellular organelles. Annual Review of Biophysics and Biomolecular Structure, 35,
199224.
Frezza, C., Zheng, L., Folger, O., Rajagopalan, K. N., MacKenzie, E. D., Jerby, L., et al.
(2011). Haem oxygenase is synthetically lethal with the tumour suppressor fumarate
hydratase. Nature, 477, 225228.
Garca-Martn, M. L., Herigault, G., Remy, C., Farion, R., Ballesteros, P., Coles, J. A., et al.
(2001). Mapping extracellular pH in rat brain gliomas in vivo by 1H magnetic resonance
spectroscopic imaging: Comparison with maps of metabolites. Cancer Research, 61,
65246531.
Gerencser, A. A., Chinopoulos, C., Birket, M. J., Jastroch, M., Vitelli, C., Nicholls, D. G.,
et al. (2012). Quantitative measurement of mitochondrial membrane potential in cultured cells: Calcium-induced de- and hyperpolarization of neuronal mitochondria. Journal of Physiology, 590, 28452871.
Gerencser, A. A., Neilson, A., Choi, S. W., Edman, U., Yadava, N., Oh, R. J., et al. (2009).
Quantitative microplate-based respirometry with correction for oxygen diffusion. Analytical Chemistry, 81, 68686878.
Gevers, W. (1977). Generation of protons by metabolic processes in heart cells. Journal of
Molecular and Cellular Cardiology, 9, 867874.
Gillies, R. J., Robey, I., & Gatenby, R. A. (2008). Causes and consequences of increased
glucose metabolism of cancers. Journal of Nuclear Medicine, 49, 24S42S.
Gimenez-Cassina, A., Garcia-Haro, L., Choi, C. S., Osundiji, M. A., Lane, E. A.,
Huang, H., et al. (2014). Regulation of hepatic energy metabolism and gluconeogenesis
by BAD. Cell Metabolism, 19, 272284.
Gimenez-Cassina, A., Martnez-Francois, J. R., Fisher, J. K., Szlyk, B., Polak, K.,
Wiwczar, J., et al. (2012). BAD-dependent regulation of fuel metabolism and
K(ATP) channel activity confers resistance to epileptic seizures. Neuron, 74, 719730.
Gubser, P. M., Bantug, G. R., Razik, L., Fischer, M., Dimeloe, S., Hoenger, G., et al.
(2013). Rapid effector function of memory CD8 + T cells requires an immediate-early
glycolytic switch. Nature Immunology, 14, 10641072.
Halestrap, A. P. (1975). The mitochondrial pyruvate carrier: Kinetics and specificity for substrates and inhibitors. Biochemical Journal, 148, 8596.
Halestrap, A. P. (2013). Monocarboxylic acid transport. Compehensive Physiology, 3,
16111643.
Helmlinger, G., Sckell, A., Dellian, M., Forbes, N. S., & Jain, R. K. (2002). Acid production
in glycolysis-impaired tumors provides new insights into tumor metabolism. Clinical
Cancer Research, 8, 12841291.
351
Hensley, C. T., Wasti, A. T., & DeBerardinis, R. J. (2013). Glutamine and cancer: Cell biology, physiology, and clinical opportunities. Journal of Clinical Investigation, 123,
36783684.
Hill, B. G., Benavides, G. A., Lancaster, J. R., Ballinger, S., DellItalia, L., Jianhua, Z., et al.
(2012). Integration of cellular bioenergetics with mitochondrial quality control and
autophagy. Biological Chemistry, 393, 14851512.
Hochachka, P. W., & Mommsen, T. P. (1983). Protons and anaerobiosis. Science, 219,
13911397.
Israelsen, W. J., Dayton, T. L., Davidson, S. M., Fiske, B. P., Hosios, A. M., Bellinger, G.,
et al. (2013). PKM2 isoform-specific deletion reveals a differential requirement for pyruvate kinase in tumor cells. Cell, 155, 397409.
Jastroch, M., Divakaruni, A. S., Mookerjee, S., Treberg, J. R., & Brand, M. D. (2010). Mitochondrial proton and electron leaks. Essays in Biochemistry, 47, 5367.
Jastroch, M., Keipert, S., & Perocchi, F. (2014). From explosives to physiological combustion: Next generation chemical uncouplers. Molecular Metabolism, 3, 8687.
Keipert, S., & Jastroch, M. J. (2014). Brite/beige fat and UCP1Is it thermogenesis?
Biochimica et Biophysica Acta - Bioenergetics, 1837, 10751082.
Kemp, G. (2005). Lactate accumulation, proton buffering, and pH change in ischemically
exercising muscle. American Journal of Physiology. Regulatory, Integrative and Comparative
Physiology, 289, R1807.
Kenwood, B. M., Weaver, J. L., Bajwa, A., Poon, I. K., Byrne, F. L., Murrow, B. A., et al.
(2014). Identification of a novel mitochondrial uncoupler that does not depolarize the
plasma membrane. Molecular Metabolism, 3, 114123.
Keuper, M., Jastroch, M., Yi, C. X., Fischer-Posovszky, P., Wabitsch, M., Tsch
op, M. H.,
et al. (2014). Spare mitochondrial respiratory capacity permits human adipocytes to
maintain ATP homeostasis under hypoglycemic conditions. FASEB Journal, 28,
761770.
Kim, H. J., & Magrane, J. (2011). Isolation and culture of neurons and astrocytes from the
mouse brain cortex. Methods in Molecular Biology, 793, 6375.
Kramer, P. A., Ravi, S., Chacko, B., Johnson, M. S., & Darley-Usmar, V. M. (2014).
A review of the mitochondrial and glycolytic metabolism in human platelets and leukocytes: Implications for their use as bioenergetic biomarkers. Redox Biology, 10, 206210.
Kushnareva, Y. E., Wiley, S. E., Ward, M. W., Andreyev, A. Y., & Murphy, A. N. (2005).
Excitotoxic injury to mitochondria isolated from cultured neurons. Journal of Biological
Chemistry, 280, 2889428902.
Liu, W., Beck, B. H., Vaidya, K. S., Nash, K. T., Feeley, K. P., Ballinger, S. W., et al. (2014).
Metastasis suppressor KISS1 seems to reverse the Warburg effect by enhancing mitochondrial biogenesis. Cancer Research, 74, 954963.
Lundholt, B. K. (2003). A simple technique for reducing edge effect in cell-based assays. Journal of Biomolecular Screening, 8, 566570.
Lunt, S. Y., & Vander Heiden, M. G. (2011). Aerobic glycolysis: Meeting the metabolic
requirements of cell proliferation. Annual Review of Cell and Developmental Biology, 27,
441464.
MacDonald, P. E., Joseph, J. W., & Rorsman, P. (2005). Glucose-sensing mechanisms in
pancreatic beta-cells. Philosophical Transactions of the Royal Society, B: Biological Sciences,
360, 22112225.
Macintyre, A. N., Gerriets, V. A., Nichols, A. G., Michalek, R. D., Rudolph, M. C.,
Deoliveira, D., et al. (2014). The glucose transporter Glut1 is selectively essential for
CD4 T cell activation and effector function. Cell Metabolism, 20, 6172.
MacIver, N. J., Michalek, R. D., & Rathmell, J. C. (2013). Metabolic regulation of
T lymphocytes. Annual Review of Immunology, 31, 259283.
352
Maddocks, O. D., Berkers, C. R., Mason, S. M., Zheng, L., Blyth, K., Gottlieb, E., et al.
(2013). Serine starvation induces stress and p53-dependent metabolic remodeling in cancer cells. Nature, 493, 542546.
Mahnensmith, R. L., & Aronson, P. S. (1985). The plasma membrane sodium-hydrogen
exchanger and its role in physiological and pathophysiological processes. Circulation
Research, 56, 773788.
Marin-Valencia, I., Yang, C., Mashimo, T., Cho, S., Baek, H., Yang, X. L., et al. (2012).
Analysis of tumor metabolism reveals mitochondrial glucose oxidation in
genetically diverse human glioblastomas in the mouse brain in vivo. Cell Metabolism,
15, 827837.
Marine, A., Krager, K. J., Aykin-Burns, N., & Macmillan-Crow, L. A. (2014). Peroxynitrite
induced mitochondrial biogenesis following MnSOD knockdown in normal rat kidney
(NRK) cells. Redox Biology, 2, 348357.
McConnell, H. M., Owicki, J. C., Parce, J. W., Miller, D. L., Baxter, G. T., Wada, H. G.,
et al. (1992). The cytosensor microphysiometer: Biological applications of silicon technology. Science, 257, 19061912.
McQuaker, S. J., Quinlan, C. L., Caldwell, S. T., Brand, M. D., & Hartley, R. C. (2013). A
prototypical small-molecule modulator uncouples mitochondria in response to endogenous hydrogen peroxide production. Chembiochem, 14, 9931000.
Metallo, C. M., Gameiro, P. A., Bell, E. L., Mattaini, K. R., Yang, J., Hiller, K., et al. (2012).
Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia. Nature,
481, 380384.
Mitchell, P. (1961). Coupling of phosphorylation to electron and hydrogen transfer by a
chemi-osmotic type of mechanism. Nature, 191, 144148.
Mouradian, M., Kikawa, K. D., Dranka, B. P., Komas, S. M., Kalyanaraman, B., &
Pardini, R. S. (2014). Docosahexaenoic acid attenuates breast cancer cell metabolism
and the Warburg phenotype by targeting bioenergetic function. Molecular Carcinogenesis.
http://dx.doi.org/10.1002/mc.22151.
Muoio, D. M., Noland, R. C., Kovalik, J. P., Seiler, S. E., Davies, M. N., DeBalsi, K. L.,
et al. (2012). Muscle-specific deletion of carnitine acetyltransferase compromises glucose
tolerance and metabolic flexibility. Cell Metabolism, 15, 764777.
Newell, K., Franchi, A., Pouyssegur, J., & Tannock, I. (1993). Studies with glycolysisdeficient cells suggest that production of lactic acid is not the only cause of tumor acidity.
Proceedings of the National Academy of Sciences of United States of America, 90, 11271131.
Newgard, C. B., An, J., Bain, J. R., Muehlbauer, M. J., Stevens, R. D., Lien, L. F., et al.
(2009). A branched-chain amino acid-related metabolic signature that differentiates
obese and lean humans and contributes to insulin resistance. Cell Metabolism, 9, 311326.
Nicholls, D. G. (2001). A history of UCP1. Biochemical Society Transactions, 29, 751755.
Nicholls, D. G. (2008). Forty years of Mitchells proton circuit: From little grey books to little
grey cells. Biochimica et Biophysica Acta - Bioenergetics, 1777, 550556.
Nicholls, D. G. (2009). Spare respiratory capacity, oxidative stress and excitotoxicity. Biochemical Society Transactions, 37, 13851388.
Nicholls, D. G., & Ferguson, S. J. (2013). Bioenergetics (4th ed.). London: Elsevier.
Nicholls, D. G., & Ward, M. W. (2000). Mitochondrial membrane potential and neuronal
glutamate excitotoxicity: Mortality and millivolts. Trends in Neuroscience, 23, 166174.
Nishiki, Y., Adewola, A., Hatanaka, M., Templin, A. T., Maier, B., & Mirmira, R. G.
(2013). Translational control of inducible nitric oxide synthase by p38 MAPK in islet
-cells. Molecular Endocrinology, 27, 336349.
Nisr, R. B., & Affourtit, C. (2014). Insulin acutely improves mitochondrial function of rat
and human skeletal muscle by increasing coupling efficiency of oxidative phosphorylation. Biochimica et Biophysica Acta, 1837, 270276.
353
Onodera, Y., Nam, J. M., & Bissell, M. J. (2014). Increased sugar uptake promotes oncogenesis via EPAC/RAP1 and O-GlcNAc pathways. Journal of Clinical Investigation, 124,
367384.
Palorini, R., Simonetto, T., Cirulli, C., & Chiaradonna, F. (2013). Mitochondrial complex
I inhibitors and forced oxidative phosphorylation synergize in inducing cancer cell death.
International Journal of Cell Biology, 243876.
Parks, S. K., Chiche, J., & Pouyssegur, J. (2013). Disrupting proton dynamics and energy
metabolism for cancer therapy. Nature Reviews Cancer, 13, 611623.
Parks, S. K., Mazure, N. M., Counillon, L., & Pouyssegur, J. (2013). Hypoxia promotes
tumor cell survival in acidic conditions by preserving ATP levels. Journal of Cell Physiology, 228, 18541862.
Parton, L. E., Ye, C. P., Coppari, R., Enriori, P. J., Choi, B., Zhang, C. Y., et al. (2007).
Glucose sensing by POMC neurons regulates glucose homeostasis and is impaired in
obesity. Nature, 449, 228232.
Perry, C. G., Kane, D. A., Lanza, I. R., & Neufer, P. D. (2013). Methods for assessing mitochondrial function in diabetes. Diabetes, 62, 10411053.
Pollizzi, K. N., & Powell, J. D. (2014). Integrating canonical and metabolic signaling
programmes in the regulation of T cell responses. Nature Reviews Immunology, 14,
435446.
Redman, E. K., Brookes, P. S., & Karcz, M. K. (2013). Role of p90RSK in regulating the
Crabtree effect: Implications for cancer. Biochemical Society Transactions, 41, 124126.
Robergs, R. A., Ghiasvanad, F., & Parker, D. (2004). Biochemistry of exercise-induced
metabolic acidosis. American Journal of Physiology. Regulatory, Integrative and Comparative
Physiology, 287, R502R516.
Robergs, R. A., & Parker, D. (2005). Lingering construct of lactic acidosis. American Journal of
Physiology. Regulatory, Integrative and Comparative Physiology, 289, R904R910.
Robinson, G. L., Dinsdale, D., Macfarlane, M., & Cain, K. (2012). Switching from aerobic
glycolysis to oxidative phosphorylation modulates the sensitivity of mantle cell lymphoma cells to TRAIL. Oncogene, 31, 49965006.
Robinson, M. M., McBryant, S. J., Tsukamoto, T., Rojas, C., Ferraris, D. V.,
Hamilton, S. K., et al. (2007). Novel mechanism of inhibition of rat kidney-type glutaminase by bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES). Biochemical Journal, 406, 407414.
Rogers, G. W., Brand, M. D., Petrosyan, S., Ashok, D., Elorza, A. A., Ferrick, D. A., et al.
(2011). High throughput microplate respiratory measurements using minimal quantities
of isolated mitochondria. PLoS One, 6, e21746.
Sakamaki, J., Fu, A., Reeks, C., Baird, S., Depatie, C., Al Azzabi, M., et al. (2014). Role of
the SIK2-p35-PJA2 complex in pancreatic -cell functional compensation. Nature Cell
Biology, 16, 234244.
Schuh, R. A., Jackson, K. C., Khairallah, R. J., Ward, C. W., & Spangenburg, E. E. (2012).
Measuring mitochondrial respiration in intact single muscle fibers. American Journal of
Physiology. Regulatory, Integrative and Comparative Physiology, 302, R712R719.
Sekine, N., Cirulli, V., Regazzi, R., Brown, L. J., Gine, E., Tamarit-Rodriguez, J., et al.
(1994). Low lactate dehydrogenase and high mitochondrial glycerol phosphate dehydrogenase in pancreatic beta-cells. Journal of Biological Chemistry, 269, 48954902.
Sukumar, M., Liu, J., Ji, Y., Subramanian, M., Crompton, J. G., Yu, Z., et al. (2013).
Inhibiting glycolytic metabolism enhances CD8 + T cell memory and antitumor function. Journal of Clinical Investigation, 123, 44794488.
Sweet, I. R., Gilbert, M., Maloney, E., Hockenberry, D. M., Schwartz, M. W., & Kim, F.
(2009). Endothelial inflammation induced by excess glucose is associated with cytosolic
glucose 6-phospate but not increased mitochondrial respiration. Diabetologia, 52, 921931.
354
Sweitach, P., Patiar, S., Supuran, C. T., Harris, A. L., & Vaughan-Jones, R. D. (2009). The
role of carbonic anhydrase 9 in regulating extracellular and intracellular pH in threedimensional tumor cell growths. Journal of Biological Chemistry, 284, 2029920310.
Tannahill, G. M., Curtis, A. M., Adamik, J., Palsson-McDermott, E. M., McGettrick, A. F.,
Goel, G., et al. (2013). Succinate is an inflammatory signal that induces IL-1 through
HIF-1. Nature, 496, 238242.
Tennant, D. A., Duran, R. V., & Gottlieb, E. (2010). Targeting metabolic transformation for
cancer therapy. Nature Reviews Cancer, 10, 267277.
Teperino, R., Amann, S., Bayer, M., McGee, S. L., Loipetzberger, A., Connor, T., et al.
(2012). Hedgehog partial agonism drives Warburg-like metabolism in muscle and brown
fat. Cell, 151, 414426.
Vaghy, P. L. (1979). Role of mitochondrial oxidative phosphorylation in the maintenance of
intracellular pH. Journal of Molecular and Cellular Cardiology, 11, 933940.
van der Windt, G. J., Everts, B., Chang, C. H., Curtis, J. D., Freitas, T. C., Amiel, E., et al.
(2012). Mitochondrial respiratory capacity is a critical regulator of CD8 + T cell memory
development. Immunity, 36, 6878.
Wang, R., Dillon, C. P., Shi, L. Z., Milasta, S., Carter, R., Finkelstein, D., et al. (2011). The
transcription factor Myc controls metabolic reprogramming upon T lymphocyte activation. Immunity, 35, 871882.
Watt, I. N., Montgomery, M. G., Runswick, M. J., Leslie, A. G., & Walker, J. E. (2010).
Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria. Proceedings of the National Academy of Sciences of United States of America, 107,
1682316827.
Weinberg, F., Hamanaka, R., Wheaton, W. W., Weinberg, S., Joseph, J., Lopez, M., et al.
(2010). Mitochondrial metabolism and ROS generation are essential for Kras-mediated
tumorigenicity. Proceedings of the National Academy of Sciences of United States of America,
107, 87888793.
Wikstrom, J. D., Sereda, S. B., Stiles, L., Elorza, A., Allister, E. M., Neilson, A., et al. (2012).
A novel high-throughput assay for islet respiration reveals uncoupling of rodent and
human islets. PLoS One, 7, e33023.
Wiley, S. E., Andreyev, A. Y., Divakaruni, A. S., Karisch, R., Perkins, G., Wall, E. A., et al.
(2013). Wolfram Syndrome protein, Miner1, regulates sulphydryl redox status, the
unfolded protein response, and Ca2+ homeostasis. EMBO Molecular Medicine, 5,
904918.
Xie, H., Valera, V. A., Merino, M. J., Amato, A. M., Signoretti, S., Linehan, W. M., et al.
(2009). LDH-A inhibition, a therapeutic strategy for treatment of hereditary leiomyomatosis and renal cell cancer. Molecular Cancer Therapeutics, 8, 626635.
Zilva, J. F. (1978). The origin of the acidosis in hyperlactataemia. Annals of Clinical Biochemistry, 15, 4043.

Seahorse

Hochgeladen von

Dokumentinformationen

Originalbeschreibung:

Copyright

Verfügbare Formate

Dieses Dokument teilen

Dokument teilen oder einbetten

Freigabeoptionen

Stufen Sie dieses Dokument als nützlich ein?

Sind diese Inhalte unangemessen?

Copyright:

Verfügbare Formate

Seahorse

Hochgeladen von

Copyright:

Verfügbare Formate

CHAPTER SIXTEEN

Analysis and Interpretation of

Seahorse Bioscience, Billerica, Massachusetts, USA

Methods in Enzymology, Volume 547

2014 Elsevier Inc.

Ajit S. Divakaruni et al.

Microplate-Based Oxygen Consumption and pH Data

handling by terminally differentiated cells (Keuper et al., 2014; Teperino

2. THE OXYGEN CONSUMPTION RATE

Ajit S. Divakaruni et al.

oxygen consumption during oxidative phosphorylation. Electrons are

Ajit S. Divakaruni et al.

consumption of the membrane potential stimulates increased activity of the

Microplate-Based Oxygen Consumption and pH Data

In some nutrient-sensing cells types, most notably pancreatic -cells

Ajit S. Divakaruni et al.

Microplate-Based Oxygen Consumption and pH Data

metabolism. Moreover, for those designing respirometry experiments to

Microplate-Based Oxygen Consumption and pH Data

2.1.5 Reserve capacity/Spare respiratory capacity

Ajit S. Divakaruni et al.

and the capacity to execute energetically demanding, cell-specific functions.

2.2. Data analysis and normalization

Microplate-Based Oxygen Consumption and pH Data

uncoupler concentration required to stimulate that rate, though this depends

Ajit S. Divakaruni et al.

consider setting up experimental groups in rows or half-columns on either

Microplate-Based Oxygen Consumption and pH Data

or automated widefield microscopy as in Affourtit et al., 2011). In their

Ajit S. Divakaruni et al.

Microplate-Based Oxygen Consumption and pH Data

increased Ca2+ cycling and mitochondrial Ca2+ overload. When normalized

Ajit S. Divakaruni et al.

effect is entirely absent, however, upon normalizing to the basal rate

2.3. Using permeabilized cells to localize bioenergetic changes

Microplate-Based Oxygen Consumption and pH Data

membrane (Diers et al., 2014). Thorough protocols and trouble-shooting

Ajit S. Divakaruni et al.

3. THE EXTRACELLULAR ACIDIFICATION RATE

Microplate-Based Oxygen Consumption and pH Data

oxidation by the rate of medium acidification in plate-based assays. As such,

Ajit S. Divakaruni et al.

3.1. Medium acidification associated with glycolytic turnover

Ajit S. Divakaruni et al.

Microplate-Based Oxygen Consumption and pH Data

Ajit S. Divakaruni et al.

An elegant proof of principle using the XF Analyzer is presented in

Microplate-Based Oxygen Consumption and pH Data

[Pi 2 ], and physiological pH) change the stoichiometry of the individual

The tight coupling between proton production and consumption breaks

Ajit S. Divakaruni et al.

Microplate-Based Oxygen Consumption and pH Data

before the development of plate-based methodologies. As an example, the

Ajit S. Divakaruni et al.

and medium pH change in response to substrates or pharmacologic agents.

Microplate-Based Oxygen Consumption and pH Data

3.2. Medium acidification not attributable to glycolytic

This may be particularly relevant in primary cells and differentiated cell

Ajit S. Divakaruni et al.

Microplate-Based Oxygen Consumption and pH Data

FCCP addition causes respiration to increase (with concomitantly high

Ajit S. Divakaruni et al.

Microplate-Based Oxygen Consumption and pH Data