CELLULAR BIOENERGETICS FOR THE 21st CENTURY

Seahorse XF Extracellular Flux Analyzers

XF Extracellular Flux Analyzer

PROVIDING A NEW WINDOW INTO CELLULAR BIOENERGETICS

Before Seahorse developed

With an understanding of cellular bioenergeticsthe processes by which cells produce and consume energyscientists 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.

the XF technology, it was difficult to measure cellular metabolismthe 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

XF ANALYZERS MEASURE THE TWO ENERGY PATHWAYS OF THE CELL

Glycolytic pathway

Oxidative phosphorylation pathway

Cells generate ATP through glycolysis, independent of oxygen to produce lactic acid. The XF Analyzer measures this process as extracellular acidification rate (ECAR).

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).

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XF Extracellular Flux Analyzer

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 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 test medium.
XF24 and XF96 Extracellular Flux Analyzers

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

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

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 islet cells 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.

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XF Extracellular Flux Analyzer

GET MORE INFORMATION AND AWARD-WINNING INNOVATION

Network ready: Transfer results to any Flexible data analysis: Excel-based analysis tools run on the instrument or on your PC. networked workstation. Solid state optical sensors: Precise, noninvasive measurement, with high sensitivity and low maintenance requirements.

Touch screen controller: Fast, intuitive, and efficient.

Robot-friendly: Compatible with robotic plate handler and fluidics systems for USB ports: Four USB ports available for maximizing connectivity and flexibility. Temperature controlled assay chamber: Compact footprint: Fits on a bench top in less than two square feet of space. Uses a standard lab power connection. Maintains normal cell physiology. Range is 7 ambient. fully automated assay protocols. Or simply load plates manually.

XF Softwareon 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.

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XF Extracellular Flux Analyzer

THE POWER OF REAL-TIME KINETICS UNLOCKS ESSENTIAL BIOENERGETIC DATA

Without oxygen measurements,
XF24 Sensor Cartridge

Seahorses 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.

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

XF96 Sensor Cartridge

OUR PATENTED TRANSIENT MICROCHAMBER MAKES IT ALL POSSIBLE

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Two or four pneumatic drug delivery ports are integrated within the sensor cartridge for sequential delivery of 25l or 75l of compound enabling dose response, agonist or antagonist response, or pathway perturbation. 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 shear on cells. 24 or 96 well tissue culture microplates support primary and cultured cells, 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.

B D C D E F F Cutaway graphic of a single probe and wall

WELL AND PROBE WORK TOGETHER TO CREATE THE TRANSIENT MICROCHAMBER

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.

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XF Extracellular Flux Analyzer

Assays of mitochondrial function are fueling our understanding of degenerative diseases and aging
Seahorse stress test reveals critical information not present in basal measurements
Being able to perform this assay in 24 or 96 wells under physiological conditions enables comprehensive experiments impossible to achieve with Clark electrode methodology.
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Seahorse bioenergetic profile of primary hippocampal neurons OCR (pMoles/min.) 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 M oligomycin to inhibit the ATP synthase; (C) 4 M FCCP, an uncoupler to short-circuit the proton circuit and allow maximal respiration; (D), a cocktail of 1M myxothiazol and 2M rotenone to inhibit total mitochondrial respiration.

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The four fundamental parameters of mitochondrial function: basal respiration, ATP turnover, proton leak, and maximal respiratory capacity using four injections per well.

XF Analyzers reveal mitochondrial dysfunction associated with oxidative stress and respiratory reserve capacity
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 g/ml oligomycin, (C) 1 M FCCP, and (D) 10 M Antimycin A to assess mitochondrial function. The nitric oxide treatment decreased the reserved capacity of the treated cells, but showed no effect on the basal oxygen consumption rate.

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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 with (B) 1 g/ml oligomycin, (C) 1 M FCCP, and (D) 10 M Antimycin A to assess mitochondrial function. Cells treated with 10 M HNE exhibit the ability overcome stress damage and exhibit normal bioenergetic reserve respiratory capacity; cell treated with 20 M HNE succumb to the stress and exhibit depleted bioenergetic reserve respiratory capacity.
OCR (% baseline) OCR (% baseline)
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[4-hydroxynonenal] for 2.5 hours. Cells were then subsequently treated

Control Control 10 M HNE 10 M HNE 20 M HNE 20 M HNE

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Data courtesy of Victor Darley-Usmar, PhD University of Alabama at Birmingham

Time (min.)

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XF Extracellular Flux Analyzer

Knowing how cells produce and use energy is essential to understanding metabolic diseases
Our label free technology reveals the kinetic effects of compounds on fatty acid oxidation without radioactivity
The 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.

Myocytes response to Metformin OCR (% Baseline) 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 M 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 M. Wu et al. GRC Molecular & Cellular Bioenergetics 2007 Poster

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Metformin + PA-BSA Metformin + Glucose Metformin + Vehicle Control + PA-BSA Control + Glucose Control + Vehicle

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XF Analyzers deliver sensitive measurement of metabolismeven in islets

OCR reveals a time-dependent increase in glucose oxidation in human islets.

Response of human pancreatic islets to glucose OCR (pMoles/min.) 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. 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, PhD Boston University Medical Center

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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

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XF Extracellular Flux Analyzer

Early detection of mitochondrial liabilities is critical to reducing attrition of new drug candidates
XF Analyzers measure dose dependent mitochondrial liabilities and lactic acidosis simultaneously in real timebefore cell viability changes
Isolating 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.

Mitochondrial impairment HepG2 liver cells exposed to increasing doses of metformin,

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ECAR (% of baseline)

buformin, or phenformin (125M.) 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.

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Metformin Buformin Phenformin

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Lactic acidosis

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Pronounced lactic acidosis, as measured by the extracellular -10 so for buformin. This is the reason that only metformin remains -30 on the market today. -40
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acidification rates -20 (ECAR), is observed with phenformin and less

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Metformin -70 Buformin Phenformin -80 Data courtesy of James Dykens, PhD & Yvonne Will, PhD

Pfizer Research -90

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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 EC50s on our drug candidatessomething that just wasnt possible before. Yvonne Will, PhD, Pfizer Inc.
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XF Extracellular Flux Analyzer

Understanding how cancer cells exploit metabolic pathways will lead to new strategies for managing cancer
XF Analyzers reveal the dependency of cancer cells on aerobic glycolysisthe Warburg effect in real time
Now 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.

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 compensation to maintain normal ATP levels. ATP was measured on the same cells after the XF analysis.

Baseline Rate & Control ATP Level

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Mitochondrial Respiration Glycolysis

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Baseline Rate & Control ATP Level

H460 tumor cells exposed to increasing concentrations of the glycolytic Glycolysis inhibitor, 2-deoxyglucose, are unable to maintain normal levels of ATP when glycolytic ATP synthesis is inhibited.
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Inhibition of aerobic glycolysis by 2DG in H460 cells

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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

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XF Extracellular Flux Analyzer

Measuring cellular bioenergetics is so easy now

XF Analyzers: Unique capabilities for measuring cell metabolism
Features
Only platform to simultaneously measure O2 consumption & H+ production rates Assays adherent cells, suspension cells, tissue sections, & subcellular components Requires less than ~104 cells per 96-well, ~5104 per 24-well Microplate format

Benefits
Measure respiration and glycolysis. Most comprehensive measure of cellular metabolism in a single assay. No longer limited to trypsinized cells. Conduct in situ analysis of live cells for a more physiologically relevant model. Uses only 1% of cells needed by other methods. You obtain more information from your precious samples. Compatible with standard assay materials, methods, & equipment so it does not interupt your work flow. Throughput 13 times that of Clark electrode. Real-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. No trypsin effects. More physiologic results. Assess up to 4 different drugs or 4 doses of a single drug with a single cell population. Provides extraordinary latitude in assay design. Eliminate label artifacts in cellular responses & drug interactions. No radioactivity, dyes, or antibodies. Use same cells in further assays. Less set-up & clean-up. Robust signal, inert, stable and accurate sensors provide superior measurements. Sensitivity better than radioactivity; easy calibration, no cleaning steps required. Facilitates better tissue culture techniques. No special clean-up needed, saves time. Easy to get started. Faster run times. Operates in normal lab environment with a standard 120 or 240 VAC power. Conserves valuable bench space. Clark electrode-type systems consume O2 and introduce artifacts into measurements.

High resolutionmeasures every 14 seconds Directly assay live attached cells Computer controlled, timed addition of up to four compounds per well Label free technology Non-flow based precision optical microsensor detection Disposable consumables Intuitive software with touch screen interface and Excel-based data analysis Compact, bench-top system

Does not consume O2

Cell types assayed on the XF Analyzer

The 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. Primary Cells (human, murine, rat, other) adipocytes, astrocytes, cardiomyocytes, cortical neurons, embryonic fibroblasts, glia, glomerular podocytes, glomeruli, hepatocytes, hippocampal neurons, neurospheres, lymphocytes, neonatal cardiomyocytes (permeabilized), proximal tubules, retinal tissue punches, smooth & striated muscle, stomach epithelia punches, tumors, umbilical vein endothelials, whole & beta pancreatic islets. Cell lines H460, A549, C2C12, HEK 293, HepG2, HUVEC, INS-1, L6, MCF7, PC12, RMS13, 3T3, SF188, MCF-7, MDA-MB-431, HCT116, Hela, PC-3, LnCAP. Other Cybrids, cybrid RhoO, CH27, A20, BW1349 b-cell hybridoma, isolated mitochondria, synaptosomes.

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. www.seahorsebio.com 9

XF Extracellular Flux Analyzer

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.

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A SEAHORSE XF ANALYZER TO FIT YOUR NEED AND BUDGET

XF24
Part number (North America, Asia) Part number (Europe) Microplate format Analyte Wells per plate Analytes per well Drug ports per well Typical wells per day Intra-well C.V. Inter-well and Inter-plate C.V. Cells per well myoblasts Cells per well rat hepatocytes Cells per well neurons Mitochondria per well Plate materials Assay running volume XF Prep Station Footprint with controller 100736-100 100737-100 24-well O2[OCR] & H+[ECAR] 24 2 4 96 <5% <20% 30-70x103 40-50x103 50-100x103 2.525g PS or PET 0.51ml/well Available 24" x 18"

XF96
100900-100 100900-101 96-well O2[OCR] & H+[ECAR] 96 2 2 480 <5% <20% 12-28x103 16-20x103 20-40x103 110g PS or PET 80200l/well Required 24" x 18"

Corporate Headquarters Seahorse Bioscience Inc. 16 Esquire Road North Billerica, MA 01862 US Phone: 1.978.671.1600 800.671.0633 www.seahorsebio.com

European Headquarters Seahorse Bioscience Europe Symbion Science Park, Boks 22 Fruebjergvej 3 2100 Copenhagen DK