CELLULAR BIOENERGETICS FOR THE 21st CENTURY

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1 CELLULAR BIOENERGETICS FOR THE 21st CENTURY Seahorse XF Extracellular Flux Analyzers

2 XF Extracellular Flux Analyzer PROVIDING A NEW WINDOW INTO CELLULAR BIOENERGETICS Before Seahorse developed 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 the XF technology, it was difficult to measure cellular metabolism the tools had been unchanged for 50 years. Now we have an instrument bioenergetics research, replacing the 50-year-old Clark electrode with the first instrument that can do in a few hours to simultaneously measure cellular respiration and glycolysis in a microplate. 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 In the presence of oxygen, glycolysis, independent of mitochondria use fatty acids oxygen to produce lactic acid. or other substrates to generate The XF Analyzer measures ATP. The XF Analyzer measures this process as extracellular this process as oxygen acidification rate (ECAR). consumption rate (OCR). 1

3 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 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 The XF Prep Station makes assay plate preparation fast, easy and error free. The Prep Station combines a non-co 2 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. 2

4 GET MORE INFORMATION AND AWARD-WINNING INNOVATION Flexible data analysis: Excel-based analysis tools run on the instrument or on your PC. Network ready: Transfer results to any networked workstation. Solid state optical sensors: Precise, noninvasive measurement, with high sensitivity and low maintenance requirements. Touch screen controller: Fast, intuitive, and efficient. USB ports: Four USB ports available for maximizing connectivity and flexibility. 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. Robot-friendly: Compatible with robotic plate handler and fluidics systems for fully automated assay protocols. Or simply load plates manually. Temperature controlled assay chamber: Maintains normal cell physiology. Range is ±7. 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. 3

5 THE POWER OF REAL-TIME KINETICS UNLOCKS ESSENTIAL BIOENERGETIC DATA Without oxygen measurements, XF24 Sensor Cartridge XF96 Sensor Cartridge 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. 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 OUR PATENTED TRANSIENT MICROCHAMBER MAKES IT ALL POSSIBLE A 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. B B 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. D C D 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. E F C E Inert optical microsensors measure O 2 consumption and H + production rates simultaneously in all wells. F 200 micron high Transient Microchamber requires a small number of cells. 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 O 2 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. 4

6 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. 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. OCR (pmoles/min.) 1000 A B C D Maximal 500 Respiration 400 Basal ATP 300 Respiration 200 Leak 100 Non-mitocondrial Respiration TIME (minutes) 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 A B C D 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. OCR (% baseline) Control Deta NO Time (min.) 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. Data courtesy of Victor Darley-Usmar, PhD University of Alabama at Birmingham OCR (% baseline) 240 A B C D Control 10 μm HNE μm HNE Time (min.) 5

7 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 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 al GRC Molecular & Cellular Bioenergetics 2007 Poster OCR (% Baseline) 160 Metformin + PA-BSA 140 Metformin + Glucose Metformin + Vehicle 120 Control + PA-BSA Control + Glucose 100 Control + Vehicle TIME (minutes) 75 XF Analyzers deliver sensitive measurement of metabolism even in islets OCR reveals a time-dependent increase in glucose oxidation in human islets. 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, PhD Boston University Medical Center OCR (pmoles/min.) mM Glucose at Control (no Glucose) +/-Glucose Oligomycin TIME (minutes) 120 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 6

8 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 time before 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, 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. OCR (% of baseline) A B C D Metformin Buformin Phenformin TIME (min.) 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. Data Courtesy of James Dykens, PhD & Yvonne Will, PhD Pfizer Research ECAR (% of baseline) A B C D Metformin Buformin Phenformin TIME (min.) 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. 7

9 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 glycolysis the 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 capacity to maintain normal ATP levels. ATP was measured on the same cells after the XF analysis. % Baseline Rate & ATP Level 180% 160% 140% 120% 100% 80% Glycolysis 60% Mitochondrial 40% OCR Respiration ECAR 20% ATP 0% Oligomycin [µm] 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. Data from Wu et al Am J Physiol 292: C125-C136, 2007 % Baseline Rate & ATP Level 180% 160% 140% 120% 100% 80% 60% 40% 20% 0% OCR ECAR ATP Mitochondrial Respiration Glycolysis Deoxyglucose [mm] 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 8

10 Measuring cellular bioenergetics is so easy now XF Analyzers: Unique capabilities for measuring cell metabolism Features Only platform to simultaneously measure O 2 consumption & H + production rates Assays adherent cells, suspension cells, tissue sections, & subcellular components Requires less than ~10 4 cells per 96-well, ~ per 24-well Microplate format High resolution measures every 14 seconds or less 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 O 2 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 O 2 and introduce artifacts into measurements. 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 islets, and pancreatic beta cells. Cell lines H460, 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. 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 for a detailed list. 9

11 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, , and on-site, support. We also provide biological application support for Seahorse Reference Assays and Applications. 10

12 A SEAHORSE XF ANALYZER TO FIT YOUR NEED AND BUDGET XF24 XF96 Part number (North America, Asia) Part number (Europe) Microplate format 24-well 96-well Analyte O 2 [OCR] & H + [ECAR] O 2 [OCR] & H + [ECAR] Wells per plate Analytes per well 2 2 Drug ports per well 4 2 Typical wells per day Intra-well C.V. <5% <5% Inter-well and Inter-plate C.V. <20% <20% Cells per well myoblasts Cells per well rat hepatocytes Cells per well neurons Mitochondria per well μg 1 10μg Plate materials PS or PET PS or PET Assay running volume 0.5 1ml/well μl/well XF Prep Station Available Required Footprint with controller 24" 18" (61cm 46cm) 24" 18" (61cm 46cm) Corporate Headquarters Seahorse Bioscience Inc. 16 Esquire Road North Billerica, MA US Phone: European Headquarters Seahorse Bioscience Europe Symbion Science Park, Boks 22 Fruebjergvej Copenhagen DK