seahorse xf - agilent.com · detailed molecular pathway mapping september 23, 2016 confidentiality...

Seahorse XF: Live Cell Metabolic Analysis

Product Specialist – XF Technology

September 23, 2016

Confidentiality Label

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Metabolic effects can be seen everywhere

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Functional metabolism complements and enriches your current data

Mass Spectroscopy

End-point Assays

Flow Cytometry

qPCR

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Respiration Glycolysis

[ H+ ]

Fermentation of • Glucose

[ Oxygen ] Oxidation of: • Glucose • Fat • Amino Acids

Metabolism in XF assays is measured via the rates of change in proton and oxygen concentration

Cells Generate Energy via Two Metabolic Pathways

XFe96 Cell Culture Microplate

XF Assay Medium

Solid State Sensor

Cell Monolayer

Sensor Probes

Fiber Optics

Well 1 Well 2

Measuring live cells in real time

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For research use only. Not for use in diagnostic procedures.

The transient microchamber enables rapid measurement of mitochondrial respiration and glycolysis in minutes

The bio-cartridge is raised, bringing the system back

to baseline

The sensor cartridge is

raised, bringing the system back

to baseline A transient

microchamber is formed

Well 1 Well 2

Measuring live cells in real time

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Drug injection ports

• Integrated, automated drug injection ports

• 4 drug injection ports per well

• Add inhibitors, stimulants, substrates, and compounds

• Injections defined by the user

Reagents in drug injector ports

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If something changed the cells energy metabolism

….Seahorse sees it! ...in real time!!

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Seahorse standardized assays measure the key metabolic pathways

XF Mito Stress Test XF Glycolysis Stress Test

XF Cell Energy Phenotype Test

XF Mito Fuel Flex Test

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XFe24 Analyzer Moderate throughput

Large well volume

(24 well)

XFe96 Analyzer Most versatile

3D biology

(96 well)

XFp Analyzer Routine and

precious samples

(8 well)

An instrument for every need and purpose

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Metabolic Engines: XF Energy Map and XF Cell Energy Phenotype Test

Define XF Metabolic Phenotypes

Glycolysis

Mitochondrial R

espiration

Aerobic

Glycolytic

Energetic

Oxyg

en

Co

nsu

mp

tio

n R

ate

(O

CR

)

Extracellular Acidification Rate

(ECAR)

XF Energy Map

• See XF Metabolic Switches

• Measure XF Metabolic Potential

Memory T Cells

Activated T cells

Neurons

Lymphoma Cells

Stressed

Neurons

Kidney Cells

Fh1-/-CL1

Kidney Cells

• Monitor Cell Quality and Health

Quiescent

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XF Assays Reveal Warburg Phenotypes that Modulate Sensitivity of Lymphoma Cells to Apoptosis

Switching from aerobic glycolysis to oxidative

phosphorylation modulates the sensitivity of mantle cell

lymphoma cells to TRAIL.

Robinson et al, Oncogene. 2012 Nov 29;31(48):4996-5006.

Control

+2DG

Z138 Mantle Cell Lymphoma Cells

+/- 2DG

Control

-Glc

Z138 Mantle Cell Lymphoma Cells

+/- glucose-free medium

TRAIL

Sensitive

TRAIL

Resistant

TRAIL= TNF-Related apoptosis Inducing Ligand

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XF Assays Reveal Metabolic Signatures in Immune Cells

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Metabolic Engines: XF Stress Tests

Measure the key parameters of

glycolytic function:

• Glycolysis

• Glycolytic Capacity

• Glycolytic Reserve

• Non-glycolytic Acidification


mitochondrial function:

• Basal respiration

• ATP coupled respiration

• Maximal Respiration

• Spare Respiratory Capacity

• Non-mitochondrial Oxygen Consumption

Gold Standard XF Assays

A B C A B C

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NEURODEGENERATION

Spare Capacity Reveals More Than Basal Metabolism 0 24

.Impaired spare respiratory capacity in cortical synaptosomes from Sod2 null mice

Flynn J M, Choi S W, Day N U, Gerencser A A, Hubbard A and Melov S., Free Radic Biol Med. 2011 Apr 1; 50(7):866-873

BASELINE OLIGO FCCP ROT / MYX

Sod2 +/+

Mouse CD-1 Synaptosomes

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Mitochondrial Standard Across Multiple Therapeutic Areas

Cell. 2014 Sep 11;158(6):1254-69.

Resetting transcription factor control circuitry toward ground-state pluripotency in human.

Takashima Y, Guo G, Loos R, Nichols J, Ficz G, Krueger F, Oxley D, Santos F,

Clarke J, Mansfield W, Reik W, Bertone P, Smith A.

Cancer Cell. 2014 Dec 8;26(6):840-50

ABL1-mediated oxidative stress adaptation in fumarate hydratase-deficient cancer

Sourbier C, Ricketts CJ, Matsumoto S, Crooks DR, Liao PJ, Mannes PZ, Yang Y, Wei MH, Srivastava

G, Ghosh S, Chen V, Vocke CD, Merino M, Srinivasan R, Krishna MC, Mitchell JB, Pendergast AM,

Rouault TA, Neckers L, Linehan WM

Cancer Cancer Cell, 2014

Immunology Science Translational Medicine, 2015

Sci Transl Med. 2015 Feb 11;7(274):274ra18

Normalization of CD4+ T cell metabolism reverses lupus.

Yin Y, Choi SC, Xu Z, Perry DJ, Seay H, Croker BP, Sobel ES, Brusko TM, Morel L.

Obesity/Diabetes Nature Medicine, 2014

Cardiovascular Disease Nature Medicine, 2014

Nat Med. 2014 Jun;20(6):616-23

Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent

stem cell and heart-on-chip technologies

Wang G, McCain ML, Yang L, He A, Pasqualini FS, Agarwal A, Yuan H, et al.

Stem Cell Biology Cell, 2014

Nat Neurosci. 2014 Dec;17(12):1710-9.

An α2-Na/K ATPase/α-adducin complex in astrocytes triggers non-cell autonomous

neurodegeneration.

Gallardo G, Barowski J, Ravits J, Siddique T, Lingrel JB, Robertson J, Steen H, Bonni A.

Neurodegeneration Nature Neuroscience, 2014

2014 Dec;20(12):1427-35.

Chronic enrichment of hepatic endoplasmic reticulum-mitochondria contact leads to

mitochondrial dysfunction in obesity

Arruda AP, Pers BM, Parlakgül G, Güney E, Inouye K, Hotamisligil GS

.

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CANCER

XF Glycolysis Stress Test Measures Glycolytic Phenotype Susceptible to Buffer Therapy

Free Base Lysine Increases Survival and

Reduces Metastasis in Prostate Cancer Model

Ibrahim-Hashim, et al., J Cancer Sci Ther 2011, S1

PC-3M

PCS

Prostate Tumor Cells

Primary Prostate Epithelial Cells

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Glycolytic Function Across Multiple Therapeutic Areas

Immunology Journal of Immunology, 2014

Autoimmunity Nature Medicine, 2015

Nat Med. 2015 Jan;21(1):55-61.

Notch controls the survival of memory CD4+ T cells by regulating glucose uptake.

Maekawa Y, Ishifune C, Tsukumo S, Hozumi K, Yagita H, Yasutomo K.

Toxicology Journal of Applied Toxicology, 2015

Cell Physiology Mitochondrion, 2015

Mitochondrion. 2015 Mar;21:19-26.

Polymerase gamma mutator mice rely on increased glycolytic flux for energy production.

Saleem A, Safdar A, Kitaoka Y, Ma X, Marquez OS, Akhtar M, Nazli A, Suri R,

Turnbull J, Tarnopolsky MA

Stem Cell Biology Cell Reports 2015

Cell Rep. 2015 Jun 16;11(10):1614-24.

mtDNA Mutagenesis Disrupts Pluripotent Stem Cell Function by Altering Redox Signaling.

Hämäläinen RH, Ahlqvist KJ, Ellonen P, Lepistö M, Logan A, Otonkoski T, Murphy

MP, Suomalainen A.

Cancer Nature, 2014

J Appl Toxicol. 2015 Aug;35(8):945-51.

Culture conditions profoundly impact phenotype in BEAS-2B, a human pulmonary epithelial

model.

Zhao F, Klimecki WT.

.

Control

Activated

Cancer Cell. 2014 Dec 8;26(6):840-50

ABL1-mediated oxidative stress adaptation in fumarate hydratase-deficient cancer

Sourbier C, Ricketts CJ, Matsumoto S, Crooks DR, Liao PJ, Mannes PZ, Yang Y, Wei MH, Srivastava

G, Ghosh S, Chen V, Vocke CD, Merino M, Srinivasan R, Krishna MC, Mitchell JB, Pendergast AM,

Rouault TA, Neckers L, Linehan WM

Nature 514, no. 7524 (2014): 628-32

Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function

Viale A, Pettazzoni P, Lyssiotis CA, Ying H, Sánchez N, Marchesini M, Carugo A, Green T, Seth

S, Giuliani V, Kost-Alimova M, Muller F, Colla S, Nezi L, Genovese G, Deem AK, Kapoor A, Yao

W, Brunetto E, Kang Y, Yuan M, Asara JM, Wang YA, Heffernan TP, Kimmelman AC, et al

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Metabolic Fuels: XF Mito Fuel Flex Test


mitochondrial fuel use:

• Dependency

• Flexibility

• Capacity

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Combining Cellular and Molecular Metabolism

Cellular Metabolism

Molecular Metabolism

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Why XF and MS?

Seahorse XF Assays Agilent Metabolomics/Flux Analyses

1. Measure rate of mitochondrial

respiration and glycolysis (big

picture)

2. Quantify rate of substrate/fuel

pathways (e.g. glucose, glutamine,

etc.)

3. Live cell metabolic rates

1. Map glycolytic and mitochondrial

pathways (detailed view)

2. Trace the flow of substrates through

metabolic pathways

3. Detailed molecular pathway

mapping

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XF assay provides live cell context; helps target and guide metabolomic assays

XF MS

XF fuel flex assay shows switch to glutamine and fatty

acids

Metabolomics and isotope tracing fully map how cells adapt to nutrient changes

Vacanti NM et al. Mol Cell. 2014 Nov 6;56(3) 425-

35.

XF substrate assay points to palmitate as key nutrient

U13C-Palmitate tracer assay explains role of fatty acids in tumor growth and survival

Caro P et al. Caner Cell. 2012 Oct

16;22(4):547-60.

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XF assays provide functional confirmation for genomic and multi-omic hits

Dendritic cell genomic screen hits multiple genes in glycolysis

Seahorse XF assay confirms

glycolytic shift required for

Dendritic cell maturation

Pantel A, et al. PLoS Biol. 2014

Jan;12(1):e1001759. doi: 10.1371

Proteomics macrophage screen identifies novel

metabolic kinase

• Glycolysis • Fructose metabolism • Mannose metabolism • Galactose metabolim • mTOR pathway • Pentose phosphate pathway (CARKL)

Top Hits

Kinase Screen

Seahorse energy map shows

novel kinase switching cell

from aerobic to anaerobic

metabolism Haschemi A er al. Cell

Metab. 2012 Jun

6;15(6):813-26.

Multi-omic XF

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Thank you

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5 µM HNE

10 µM HNE

HNE

0 µM HNE

Oligo

FCCP Antimycin A

0

50

100

150

250

300

0 25 50 75 100 125 150 175 200

OC

R (

% b

aselin

e)

Time (min)

200

Importance of the bioenergetic

reserve capacity in response to

cardiomyocyte stress induced by 4-

hydroxynonenal

Bradford G. Hill, Brian P. Dranka,

Luyun Zou, John C. Chatham and

Victor M. Darley-Usmar, Biochem J.

2009 Nov 15;425(1):99-107

CARDIOVASCULAR XF Stress Test Sensitivity Reveals Sub-Lethal Stress Response and Mechanism

XF Cell Mito Stress Test

20 µM HNE

Neonatal Rat Ventricular Myocytes

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seahorse xf - agilent.com · detailed molecular pathway mapping september 23, 2016 confidentiality...

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