Seahorse Metabolic Analyzers Enable Rapid, Quantitative Measurement of Cellular Bioenergetics
David Ferrick, PhD Chief Scientific Officer
Changing the Way Metabolism is Measured Mitochondria
Energy Map
Glycolysis
Fatty Acids
Glucose Glutamine
O 2 O 2
O 2
O 2
O 2
O 2
+ H + H
+ H
+ H + H H +
O 2 O 2
O 2
Seahorse Stressor Reagent (oligomycin & FCCP)
Let’s start with the most rapid, user friendly measure of bioenergetic behavior: The XF Cell Energy Phenotype Test
Sensor Cartridge and miniPlate with your cells
Your XFp
Measure any metabolic change, in any cell, for any cause, with the XF Energy Map
Glycolysis
Mito
chon
dria
l Res
pira
tion
Aerobic
Glycolytic
Energetic
Oxy
gen
Cons
umpt
ion
Rate
(OCR
)
Extracellular Acidification Rate (ECAR)
XF Energy Map
Quiescent
Define metabolic phenotypes
Glycolysis
Mito
chon
dria
l Res
pira
tion
Aerobic
Glycolytic
Energetic
Oxy
gen
Cons
umpt
ion
Rate
(OCR
)
Extracellular Acidification Rate (ECAR)
XF Energy Map
Quiescent
Neurons
Activated T cells
Memory T Cells Tumor Cells
Measure metabolic potential
Glycolysis
Mito
chon
dria
l Res
pira
tion
Aerobic
Glycolytic
Energetic
Oxy
gen
Cons
umpt
ion
Rate
(OCR
)
Extracellular Acidification Rate (ECAR)
XF Energy Map
Neurons
Stressed Neurons
Quiescent
Stressed
Impaired Neurons
Stressed
At risk Neurons
Watch cells switch metabolic pathways
Glycolysis
Mito
chon
dria
l Res
pira
tion
Aerobic
Glycolytic
Energetic
Oxy
gen
Cons
umpt
ion
Rate
(OCR
)
Extracellular Acidification Rate (ECAR)
XF Energy Map
Tumor Cells
Quiescent
Stressed
p53 Arrested tumor Cell
Stressed
Energy map reveals three metabolic phenotypes regulating Mycobacterium tuberculosis drug susceptibility
Drug Sensitive
Ergothioneine Maintains Redox and Bioenergetic Homeostasis Essential for Drug Susceptibility and Virulence of Mycobacterium tuberculosis. Saini et al., 2016, Cell Reports 14, 1–14, January 26, 2016
Metabolic reprogramming regulates stem cell differentiation
Reprogramming of Stem Cell Epigenetics. Ryall JG, Cliff T, Dalton S, Sartorelli V Cell Stem Cell. 2015 Dec;3;17(6):651-62.
Changes in metabolism regulate many of the important cell fate decisions made by stem cells
Seahorse standardized assays measure the key metabolic pathways
XF Mito Stress Test XF Glycolysis Stress Test
XF Cell Energy Phenotype Test
Metabolic Engines: XF Stress Test Kits
Measure the key parameters of glycolytic function:
• Glycolysis • Glycolytic Capacity • Glycolytic Reserve • Non-glycolytic Acidification
Extr
acel
lula
r Aci
dific
atio
n
EC
AR
(mpH
/min
)
Non-glycolytic Acidification
Glycolytic Capacity
Glycolysis
Glycolytic Reserve Capacity
Measure the key parameters of mitochondrial function:
• Basal respiration • ATP coupled respiration • Maximal Respiration • Spare Respiratory Capacity • Non-mitochondrial Oxygen Consumption
XF Mito Stress Test XF Glycolysis Stress Test A B C A B C
Gold Standard XF Assays
Mitochondrial Function 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
.
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
Seahorse mito stress test reveals requirement of fatty acid oxidation to develop immune memory
Key discovery in immune metabolism may improve immunotherapy
+ FA
Immunity 2011 December 28 Washington University School of Medicine, St. Louis
Durability of engineered patient T cells may depend on triggering the appropriate metabolic pathways
“The implications of Prof. Pearce’s findings may help explain the dramatic clinical successes of cancer
immunotherapies”, Dr. Carl June
- FA
Memory T Cell
Memory failure
Naive T cell Activate T cell
Effector T cell
Memory T cell
Metabolic programs control immune cell differentiation and function
Aerobic Energetic
Glycolytic Quiescent
Aerobic Energetic
Glycolytic Quiescent
Aerobic Energetic
Glycolytic Quiescent
GSEA (Gene set enrichment analysis) identifies metabolic program for Dendritic Cell maturation
Direct type I IFN but not MDA5/TLR3 activation of dendritic cells is required for maturation and metabolic shift to glycolysis after poly IC stimulation. Pantel A et al. PLoS Biol. 2014 Jan;12(1):e1001759.
Monocyte / resident macrophage M1 macrophage M2 macrophage
Metabolic programs control immune cell differentiation and function
Aerobic Energetic
Glycolytic Quiescent
Aerobic Energetic
Glycolytic Quiescent
Aerobic Energetic
Glycolytic Quiescent
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
Metabolic Fuels: XF Mito Fuel Flex Test
Measure the key parameters of mitochondrial fuel use:
• Dependency
• Flexibility
• Capacity
Glucose Pathway
Flexibility
Dependency
Capacity
100
80
60
40
20
0 Glutamine Pathway
80
60
40
20
0
Flexibility
Dependency
Capacity
100
Fatty Acid Pathway
Flexibility
80
60
40
20
0
Dependency
Capacity
100
- The Newest Standard for Live Cell Metabolism
This unique substrate assay is highly complimentary to other substrate utilization assays
2-NBDG
+ Live cell rate + Real-time + Fuel Dependency + Fuel Flexibility + Fuel Capacity
= XF Mito Fuel Flex Test
13C Glucose Flux Analysis 2-NBDG Glucose uptake
[14C] 2-DG uptake Glucose consumption
XF Mito Fuel Flex Test
The Agilent-Seahorse Advantage
Metabolic signatures uncover distinct targets in molecular subsets of diffuse large B cell lymphoma Caro P, et al, Cancer Cell; 2012 Oct 16. 22 (4):547-560.
- Seahorse Assays inform 13C isotopomer analyses
How do cells survive without a functional TCA cycle in HLRCC (Hereditary leiomyomatosis and renal-cell cancer)
Aerobic Glycolysis
Lactate
Haem oxygenase is synthetically lethal with the tumour suppressor fumarate hydratase. Frezza C et al. Nature. 2011 Aug 17;477(7363):225-8.
Computational model predicts novel pathway that is synthetic lethal for Fh1-/-
fumarate
succinate
Haem
18/24 reactions predicted to be synthetic lethal belonged to haem metabolism
Confirmed by metabolomics
Haem oxygenase is synthetically lethal with the tumour suppressor fumarate hydratase. Frezza C et al. Nature. 2011 Aug 17;477(7363):225-8.
Potential new approach for treating HLRCC patients
Synthetic lethal (in silico) Upregulated in Fh-/-
Substrates induced in Fh-/-
bilirubin
bilirubin-UDP
fumarate
succinate
glutamine
Lactate
Haem
Bilirubin-UDP
bilirubin
Haem oxygenase is synthetically lethal with the tumour suppressor fumarate hydratase. Frezza C et al. Nature. 2011 Aug 17;477(7363):225-8.
Seahorse provides metabolic information on key pathways in living cells
Mitochondrial function
Glycolytic function
Metabolic phenotype
Glucose
Glutamine
Fatty acids
ENGINES
FUELS
Next Generation Metabolism
Questions?
THANK YOU!
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