cellular bioenergetics for the 21st centurybosch.test.med.usyd.edu.au/facilities/molecular... ·...

XF Extracellular Flux Analyzer

Seahorse XF Extracellular Flux Analyzers

CELLULAR BIOENERGETICSFOR THE 21st CENTURY


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



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



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.



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



Assays of mitochondrial function are fueling our understanding of degenerative diseases and aging

60 70 80 90 10050403020100

1000

900

800

700

600

500

400

300

200

100

0

TIME (minutes)

OC

R (p

Mo

les/

min

.)Basal

Respiration

Leak

ATP

Maximal

Respiration

BA C D

Non-mitocondrial Respiration

0

30

60

120

180

90

150

210

240

OC

R (%

bas

elin

e)

Time (min.)

20 μM HNE

Control10 μM HNE

0 200175150125100755025

A B C D

OC

R (%

bas

elin

e)

Time (min.)150135120105453015

Deta NOControl

100

-100

-80

-60

-40

-20

0

20

40

60

80

A B C D

0

30

60

120

180

90

150

210

240

OC

R (%

bas

elin

e)

Time (min.)

20 μM HNE

Control10 μM HNE

0 200175150125100755025

A B C D

OC

R (%

bas

elin

e)

Time (min.)150135120105453015

Deta NOControl

100

-100

-80

-60

-40

-20

0

20

40

60

80

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



Knowing how cells produce and use energy is essential to understanding metabolic diseases

750 15 30 45 60

160

-20

0

20

40

60

80

100

120

140

OC

R (%

Bas

elin

e)

TIME (minutes)

Metformin + PA-BSAMetformin + GlucoseMetformin + VehicleControl + PA-BSAControl + GlucoseControl + Vehicle

TIME (minutes)

OC

R (p

Mo

les/

min

.)

120

800

700

600

500

400

300

20012 24 36 48 60 72 84 96 108

+/-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



Early detection of mitochondrial liabilities is critical to reducing attrition of new drug candidates

35

30

25

20

15

10

5

0

-520 30 40 50 60 70 80 90 100 110

TIME (min.)

EC

AR

(% o

f b

asel

ine)

10

-20

-10

0

-30

-40

-50

-60

-70

-80

-9020 30 40 50 60 70 80 90 100 110

TIME (min.)

OC

R (%

of

bas

elin

e)

Phenformin

MetforminBuformin

A B C D

Phenformin

MetforminBuformin

A B C D

35

30

25

20

15

10

5

0

-520 30 40 50 60 70 80 90 100 110

TIME (min.)

EC

AR

(% o

f b

asel

ine)

10

-20

-10

0

-30

-40

-50

-60

-70

-80

-9020 30 40 50 60 70 80 90 100 110

TIME (min.)

OC

R (%

of

bas

elin

e)

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.



Understanding how cancer cells exploit metabolic pathways will lead to new strategies for managing cancer

0%

20%

40%

60%

80%

100%

120%

140%

180%

160%

0 0.008 0.04 0.2 1 5

% B

asel

ine

Rat

e &

AT

P L

evel

Oligomycin [µM]

ATP

OCRECAR

% B

asel

ine

Rat

e &

AT

P L

evel

0%

40%

100%

140%

180%

20%

60%

80%

120%

160%

0 0.8 4 20 50 100 200

2-Deoxyglucose [mM]

ATP

OCRECAR

Glycolysis

MitochondrialRespiration


Glycolysis

0%

20%

40%

60%

80%

100%

120%

140%

180%

160%

0 0.008 0.04 0.2 1 5

% B

asel

ine

Rat

e &

AT

P L

evel

Oligomycin [µM]

ATP

OCRECAR

% B

asel

ine

Rat

e &

AT

P L

evel

0%

40%

100%

140%

180%

20%

60%

80%

120%

160%

0 0.8 4 20 50 100 200

2-Deoxyglucose [mM]

ATP

OCRECAR

Glycolysis



Glycolysis

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



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.



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

cellular bioenergetics for the 21st centurybosch.test.med.usyd.edu.au/facilities/molecular... ·...

Documents