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XF Extracellular Flux Analyzer
Seahorse XF Extracellular Flux Analyzers
CELLULAR BIOENERGETICSFOR THE 21st CENTURY
XF Extracellular Flux Analyzer
www.seahorsebio.com 1
PROVIDING A NEW WINDOW INTO CELLULAR BIOENERGETICS
“ Before Seahorse developed
the XF technology, it was
difficult to measure cellular
metabolism—the tools had
been unchanged for 50 years.
Now we have an instrument
that can do in a few hours
what once took days.”
— John Lemasters, Medical University of South Carolina
With an understanding of cellular bioenergetics—the processes by which cells
produce and consume energy—scientists are connecting genomic and proteomic
data to physiologic traits of cells and generating new insights into obesity, diabetes,
cancer, cardiovascular and neurodegenerative diseases.
Seahorse XF Extracellular Flux Analyzers are the driving force behind the new cellular
bioenergetics research, replacing the 50-year-old Clark electrode with the first instrument
to simultaneously measure cellular respiration and glycolysis in a microplate.
XF ANALYZERS MEASURE THE TWO ENERGY PATHWAYS OF THE CELL
In the presence of oxygen,
mitochondria use fatty acids
or other substrates to generate
ATP. The XF Analyzer measures
this process as oxygen
consumption rate (OCR).
Cells generate ATP through
glycolysis, independent of
oxygen to produce lactic acid.
The XF Analyzer measures
this process as extracellular
acidification rate (ECAR).
Oxidative phosphorylation pathway
Glycolytic pathway
XF Extracellular Flux Analyzer
www.seahorsebio.com 2
DESIGNED BY SCIENTISTS FOR SCIENTISTS
The Seahorse XF Extracellular Flux Analyzer makes cellular bioenergetic studies simple, efficient, and user-friendly. Designed in collaboration with bioenergetics experts in academia, pharma, and biotech, XF Analyzers provide ease-of-use and throughput that is far superior to other methods.
XF Analyzer: 24-well and 96-well format
The XF Analyzer simultaneously measures mitochondrial respiration and glycolysis in cells in minutes, using label-free disposable optical sensors. This versatile bench top instrument takes little room; assays primary and adherent cells; tumor and suspension cells; islets of Langerhans and isolated mitochondria. XF Analyzers provide the most physiologically relevant in vitro measurement for your bioenergetics studies.
XF Prep Station: 24-well and 96-well format
The XF Prep Station makes assay plate preparation fast, easy and error free. The Prep Station combines a non-CO2 incubator and an automated medium changer that improves reproducibility and simplifies changes from growth medium to low buffered assay medium. It also provides integrated control via the XF instrument touch screen software.
XF Consumables
Seahorse designs and manufactures all the consumable components critical to the success of your bioenergetic assays. All you need are your cells and compounds.
FluxPaks: Includes XF assay kits with disposable sensor cartridges, cell culture microplates, calibration plates, and XF calibrant.
Cell Culture Microplates: Designed to maximize the sensitivity of the sensor cartridge and allow for repeated measurements. Tissue culture treated for optimum adherence and growth of mammalian cells.
Islet Capture Microplates: Created specifically to assay the cellular metabolism of islets of Langerhans with an XF24 Analyzer, keeps islets an optimal distance from the XF sensor while allowing for the free exchange of nutrients and gasses to maintain islets healthy throughout the assay. (XF96 version in development)
XF Calibrant: Premixed and ready-to-use.
XF Assay Medium: A specially formulated glucose-free, bicarbonate-free, DMEM providing convenience while enhancing XF data quality.
XF24 and XF96 Extracellular Flux Analyzers
“ I trust Seahorse technology.
The XF allows us to analyze
the bioenergetics of cells
under normal growth
conditions with throughput
that is astounding.”
— Martin Brand, Buck Institute & MCR Dunn
XF Extracellular Flux Analyzer
www.seahorsebio.com 3
GET MORE INFORMATION AND AWARD-WINNING INNOVATION
Flexible data analysis:
Excel-based analysis
tools run on the instrument
or on your PC.
Touch screen controller:
Fast, intuitive, and efficient.
Compact footprint:
Fits on a bench top in less than
two square feet (0.2 square meters)
of space. Uses a standard lab
power connection.
Temperature controlled assay chamber:
Maintains normal cell physiology.
Range is ±7°.
Robot-friendly:
Compatible with robotic plate
handler and fluidics systems for
fully automated assay protocols.
Or simply load plates manually.
Solid state optical sensors:
Precise, noninvasive measurement,
with high sensitivity and low
maintenance requirements.
Network ready:
Transfer results to any
networked workstation.
XF Software—on your XF Analyzer and your computers
XF Software simplifies your bioenergetics experimental design and analysis. The intuitive Excel-based system is remarkably flexible and user friendly, letting you manage experiments, acquire data and display results to your specifications with easy-to-use XF Assay templates. You can design experiments with the touch screen controller or from the convenience of your own computer. You can also access data in real time or store your experimental conditions and results in an Excel file for later analysis.
USB ports:
Four USB ports available
for maximizing connectivity
and flexibility.
XF Extracellular Flux Analyzer
www.seahorsebio.com 4
THE POWER OF REAL-TIME KINETICS UNLOCKS ESSENTIAL BIOENERGETIC DATA
“ Without oxygen measurements,
cellular bioenergetics would
be in the dark ages. If I had
the Seahorse 30 years ago,
I would have accomplished
so much more.”
— David Nichols, Buck Institute & Lund University
Seahorse’s patented Transient Microchamber is the key to
measuring real-time changes in minutes rather than hours. The
Transient Microchamber enables you to perform precise, label free,
nondestructive measurements on 24 or 96 samples simultaneously,
with automated, sequential addition of up to four drugs. Because
XF measurements are nondestructive, the metabolic rate of the
same cell population can be measured repeatedly over time.
B
CE
F
D
A A
Two or four pneumatic drug delivery ports are integrated within the sensor cartridge for sequential delivery of 25μl or 75μl of compound enabling dose response, agonist or antagonist response, or pathway perturbation, etc.
Vertically lowering sensor probes gently create a Transient Microchamber, allowing rapid, real-time measurement of changes in oxygen and proton concentrations in the medium proximal cells, to simultaneously measure their rates of oxidation and glycolytic metabolism.
Chamber wall is designed to eliminate cell sheer.
24 or 96 well tissue culture microplates support primary cells and cell lines; yeast; tissue and subcellular components.
Inert optical microsensors measure O2 consumption and H+ production rates simultaneously in all wells.
200 micron high Transient Microchamber requires a small number of cells.
OUR PATENTED TRANSIENT MICROCHAMBER MAKES IT ALL POSSIBLE
Cutaway graphic of a single probe and wall
WELL AND PROBE WORK TOGETHER TO CREATE THE TRANSIENT MICROCHAMBER
XF96 Sensor Cartridge
XF24 Sensor Cartridge
Sensors lower to form
Transient Microchambers
enabling rapid, real-time
kinetic measurement of
O2 consumption and
H+ production rates in
live cells.
Sensors in the raised position
allow medium to re-equilibrate,
restoring cells to baseline
and enabling repeated
measurement of cells.
A
B
C
D
E
F
XF Extracellular Flux Analyzer
www.seahorsebio.com 5
Assays of mitochondrial function are fueling our understanding of degenerative diseases and aging
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TIME (minutes)
OC
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.)Basal
Respiration
Leak
ATP
Maximal
Respiration
BA C D
Non-mitocondrial Respiration
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20 μM HNE
Control10 μM HNE
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Deta NOControl
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A B C D
Seahorse bioenergetic profile of primary hippocampal neurons
After measuring the basal respiration rate of primary hippocampal
neurons, compounds modulating mitochondrial function were
added sequentially into the assay medium. The effect on oxygen
consumption rates (OCR) was measured after each compound
addition. (A) control medium; (B) 1.2 μM oligomycin to inhibit the
ATP synthase; (C) 4 μM FCCP, an uncoupler to short-circuit the proton
circuit and allow maximal respiration, also known as reserve capacity;
(D) a cocktail of 1μM myxothiazol and 2μM rotenone to inhibit total
mitochondrial respiration.
Dysfunctional respiratory capacity not detected in basal respiration rates
Bovine aortic endothelial cells were stressed by exposure to (A) NO [(z)-1-
[2-(2-Aminoethyl)-N-(2ammonioethyl)amino]diazen-1-ium-1,2-diolate] for
2 hours. Treated and control cells were then subsequently treated with (B)
1 μg/ml oligomycin, (C) 1 μM FCCP, and (D) 10 μM Antimycin A to assess
mitochondrial function. The nitric oxide treatment decreased the reserve
capacity of the uncoupled cells, but showed no effect on the basal oxygen
consumption rate.
Oxidative stress impact on bioenergetic reserve capacity
Neonatal rat ventricular myocyte primary cells were exposed to
pathologically relevant concentrations of the reactive lipid species (A) HNE
[4-hydroxynonenal] for 2.5 hours. Cells were then subsequently treated
with (B) 1 μg/ml oligomycin, (C) 1 μM FCCP, and (D) 10 μM Antimycin A to
assess mitochondrial function. Cells treated with 10 μM HNE exhibit the ability
overcome stress damage and exhibit normal bioenergetic reserve respiratory
capacity; cell treated with 20 μM HNE succumb to the stress and exhibit
depleted bioenergetic reserve respiratory capacity.
Seahorse stress test reveals critical information not present in basal measurementsBeing able to perform this assay in 24 or 96 wells under physiological conditions enables comprehensive experiments
impossible to achieve with Clark electrode methodology.
XF Analyzers reveal mitochondrial dysfunction associated with oxidative stress and respiratory reserve capacity
The four fundamental parameters of mitochondrial function: basal respiration, ATP turnover, proton leak, and maximal respiratory capacity using four injections per well.
Data courtesy of Victor Darley-Usmar, PhDUniversity of Alabama at Birmingham
XF Extracellular Flux Analyzer
www.seahorsebio.com 6
Knowing how cells produce and use energy is essential to understanding metabolic diseases
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Metformin + PA-BSAMetformin + GlucoseMetformin + VehicleControl + PA-BSAControl + GlucoseControl + Vehicle
TIME (minutes)
OC
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min
.)
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+/-Glucose Oligomycin
20mM Glucose at
Control (no Glucose)
Myocytes response to Metformin
C2C12 Myocyte cultures pretreated for 24 hours with 2 mM metformin
or vehicle control were injected with fatty acid and glucose to final
concentrations of 150 μM and 25 mM respectively. Palmitate
stimulated the oxygen consumption rates (OCR) in metformin-treated
cells, while glucose did not. Both palmitate and glucose stimulated
OCR in control cells suggesting selective oxidation of palmitate over
glucose in metformin-treated cells.
Data from Wu et alGRC Molecular & Cellular Bioenergetics 2007 Poster
Response of human pancreatic islets to glucose
Glucose injection increases the OCR of pancreatic islets over the basal
respiration rate, which has been shown to correlate to insulin secretion.
Response to glucose is blunted in diabetic islets (data not shown.)
Sequential addition of the ATP synthase inhibitor oligomycin shows the
mitochondrial coupling efficiency or ATP turnover of the islets.
Data courtesy of Orian Shirihai, MD, PhDBoston University Medical Center
Our label free technology reveals the kinetic effects of compounds on fatty acid oxidation— without radioactivityThe multiple drug ports and wells allow eloquent experiments to be performed on the same cells in one microplate. Another
example of the cellular bioenergetic-revealing experiments that cannot be achieved with any other technology.
XF Analyzers deliver sensitive measurement of metabolism—even in isletsOCR reveals a time-dependent increase in glucose oxidation in human islets.
“ Our Seahorse has been essential in our elucidation of the function
of mitochondrial genes and proteins, establishing a link between
mitochondrial function and type-2 diabetes.”
—Vamsi Mootha, Massachusetts General Hospital
XF Extracellular Flux Analyzer
www.seahorsebio.com 7
Early detection of mitochondrial liabilities is critical to reducing attrition of new drug candidates
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Phenformin
MetforminBuformin
A B C D
Phenformin
MetforminBuformin
A B C D
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Phenformin
MetforminBuformin
A B C D
Phenformin
MetforminBuformin
A B C D
Mitochondrial impairment
HepG2 liver cells exposed to increasing doses of metformin,
buformin, or phenformin (125μM.) A clear and marked dose
dependent decrease in mitochondrial respiration, as measured
by the oxygen consumption rates (OCR), is observed with
phenformin being the most potent.
Lactic acidosis
Pronounced lactic acidosis, as measured by the extracellular
acidification rates (ECAR), is observed with phenformin and less
so for buformin. This is the reason that only metformin remains
on the market today.
XF Analyzers measure dose dependent mitochondrial liabilities and lactic acidosis simultaneously in real time—before cell viability changesIsolating mitochondria has been an obstacle to implementing routine mitochondrial safety testing. With Seahorse cellular bioenergetic
measurements this information in mitochondria or cells is easily obtainable and the additional parameter of glycolysis provides critical
information unavailable from any other mitochondrial assay.
Data Courtesy of James Dykens, PhD & Yvonne Will, PhDPfizer Research
“ The XF96 Analyzer has revolutionized the way we approach toxicity screening.
Since getting the high throughput capabilities of the XF96 Analyzer, we routinely
generate 6 point cellular bioenergetic EC50’s on our drug candidates—something
that just wasn’t possible before.”
—Yvonne Will, PhD, Pfizer Inc.
XF Extracellular Flux Analyzer
www.seahorsebio.com 8
Understanding how cancer cells exploit metabolic pathways will lead to new strategies for managing cancer
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Inhibition of mitochondrial respiration by oligomycin in H460 cells
H460 tumor cells exposed to increasing concentrations of the
complex V inhibitor, oligomycin, show sufficient glycolytic capacity to
maintain normal ATP levels. ATP was measured on the same cells
after the XF analysis.
Inhibition of aerobic glycolysis by 2DG in H460 cells
H460 tumor cells exposed to increasing concentrations of the
glycolytic inhibitor, 2-deoxyglucose, are unable to maintain normal
levels of ATP when glycolytic ATP synthesis is inhibited due to a
compromised OXPHOS system.
XF Analyzers reveal the dependency of cancer cells on aerobic glycolysis—the Warburg effect—in real timeNow in a microplate format you can study how manipulating OXPHOS, glycolysis, and glucose and glutamine dependencies
associated with cancer, can aid in developing new drugs to understand and fight cancer.
XF Assays show the bioenergetic plasticity of small cell lung carcinoma cells. The glycolysis rate of H460 cells elevates to compensate
for the inhibition of mitochondrial oxidative phosphorylation and cells successfully maintain cellular ATP levels. However, while the
respiration rate elevates to compensate for the inhibition of glycolysis, the cells fail to sustain the cellular ATP levels. This data indicates
that H460 cells depend upon aerobic glycolysis or to meet their energy demand.
Data from Wu et al Am J Physiol 292: C125-C136, 2007
“ My XF Analyzer has transformed my investigations on the regulation of
cancer metabolism by oncogenes and on the role they play in oncogenesis.”
—Ben Van Houten, University of Pittsburgh Cancer Institute
XF Extracellular Flux Analyzer
www.seahorsebio.com 9
More than 100 cell lines from most source tissue and species have been used with the XF Analyzer. Visit www.seahorsebio.com for a detailed list.
Measuring cellular bioenergetics is so easy now
XF Analyzers: Unique capabilities for measuring cell metabolism
Features Benefits
Only platform to simultaneously measure O2 consumption & H+ production rates
Measure respiration and glycolysis. Most comprehensive measure of cellular metabolism in a single assay.
Assays adherent cells, suspension cells, tissue sections, & subcellular components
No longer limited to trypsinized cells. Conduct in situ analysis of live cells for a more physiologically relevant model.
Requires less than ~104 cells per 96-well, ~5×104 per 24-well
Uses only 1% of cells needed by other methods. You obtain more information from your precious samples.
Microplate formatCompatible with standard assay materials, methods, & equipment so it does not interupt your work flow. Throughput 13 times that of Clark electrode.
High resolution—measures every 14 seconds or lessReal-time kinetic results in minutes, not hours and > 95% faster than other methods.Measure a single population of cells over a period of minutes, hours, or days.
Directly assay live attached cells No trypsin effects. More physiologic results.
Computer controlled, timed addition of up to four compounds per well
Assess up to 4 different drugs or 4 doses of a single drug with a single cell population. Provides extraordinary latitude in assay design.
Label free technologyEliminate label artifacts in cellular responses & drug interactions. No radioactivity, dyes, or antibodies. Use same cells in further assays. Less set-up & clean-up.
Non-flow based precision optical microsensor detectionRobust signal, inert, stable and accurate sensors provide superior measurements.Sensitivity better than radioactivity; easy calibration, no cleaning steps required.
Disposable consumables Facilitates better tissue culture techniques. No special clean-up needed, saves time.
Intuitive software with touch screen interface and Excel-based data analysis
Easy to get started. Faster run times.
Compact, bench-top systemOperates in normal lab environment with a standard 120 or 240 VAC power.Conserves valuable bench space.
Does not consume O2
Clark electrode-type systems consume O2 and introduce artifacts into measurements.
Primary Cells (human, murine, rat, other)adipocytes, astrocytes, cardiomyocytes, cortical neurons, embryonic fibroblasts, glia, glomerular podocytes, glomeruli, hepatocytes, hip-pocampal neurons, neurospheres, lymphocytes, neonatal cardio-myocytes (permeabilized), proximal tubules, retinal tissue punches, smooth & striated muscle, stomach epithelia punches, tumors, umbili-cal vein endothelials, whole islets, and pancreatic beta cells.
Cell linesH460, A549, C2C12, HEK 293, HepG2, HUVEC, INS-1, L6, MCF7, PC-12, RMS13, 3T3, SF188, MCF-7, MDA-MB-431, HCT116, Hela, PC-3, LnCAP.
OtherCybrids, cybrid RhoO, CH27, A20, BW1349 b-cell hybridoma, isolated mitochondria, synaptosomes.
Cell types assayed on the XF AnalyzerThe XF Analyzer has been used with a large variety of primary cells and cell lines. Listed below is a selection of common cells used with the XF Analyzer.
XF Extracellular Flux Analyzer
www.seahorsebio.com 10
SEAHORSE TEAM OF EXPERTS SUPPORT YOUR WORK
“ The expert scientific support
my lab received from the
Seahorse Team allowed us
to develop a new screening
assay in just a few months.”
— Irene Edebert, AstraZeneca, Sweden
XF Training
XF Training is available for new and experienced XF users, as well as for those considering the purchase of an XF instrument. Our hands-on training programs cover the use of the instrument and software and takes you from a review of cellular bioenergetics to general XF assay development, through data analysis and graphics. More advanced application and assay specific training is also available. Seahorse Bioscience is committed to ensuring your success.
The XF Support Team
The XF Support Team includes a worldwide customer support organization dedicated to helping customers perform productive assays that consistently produce high quality data. Our Support Team performs field installations, annual preventative maintenance checks, repairs, and software updates as well as phone, email, and on-site, support. We also provide biological application support for Seahorse Reference Assays and Applications.
A SEAHORSE XF ANALYZER TO FIT YOUR NEED AND BUDGET
XF24 XF96
Part number (North America, Asia) 100736-100 100900-100
Part number (Europe) 100737-100 100900-101
Microplate format 24-well 96-well
Analyte O2[OCR] & H+[ECAR] O2[OCR] & H+[ECAR]
Wells per plate 24 96
Analytes per well 2 2
Drug ports per well 4 2
Typical wells per day 96 480
Intra-well C.V. <5% <5%
Inter-well and Inter-plate C.V. <20% <20%
Cells per well – myoblasts 30-70×103 12-28×103
Cells per well – rat hepatocytes 40-50×103 16-20×103
Cells per well – neurons 50-100×103 20-40×103
Mitochondria per well 2.5–25μg 1–10μg
Plate materials PS or PET PS or PET
Assay running volume 0.5–1ml/well 80–200μl/well
XF Prep Station Available Required
Footprint with controller 24"×18" (61cm×46cm) 24"×18" (61cm×46cm)
Corporate Headquarters
Seahorse Bioscience Inc.16 Esquire RoadNorth Billerica, MA 01862 USPhone: 1.978.671.1600 • 800.671.0633
www.seahorsebio.com
European Headquarters
Seahorse Bioscience EuropeSymbion Science Park, Boks 22Fruebjergvej 32100 Copenhagen DK