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For Research Use Only. Not for diagnostic purposes.
Daniel Disher
Manager, Seahorse XF Instrument Sales
Midwest and West Coast
Baseline
Stressed
Glycolytic
Aerobic Energetic
Quiescent
For Research Use Only. Not for diagnostic purposes.
Metabolic effects can be seen everywhere
July 11, 2017
Confidentiality Label
2
Seahorse is the most used, cited measure of functional metabolism
>1,300 instruments >10,000 users
>2,400 publications (Jan 2016)
232
409
600
783
1 7 16 41 103
240
472
881
1,481
2,264
0
500
1000
1500
2000
2500
Publications Cumulative
Energy metabolism is a leader not a follower: therefore reprograming metabolism may alter outcomes
Proliferation
Normal
Tumor cells
Novel cancer
therapies
Death
Healthy
neurons
Dying
neurons
Neurodegeneration
Activation
Naive
immune cells
Effector & memory
immune cells
Vaccines &
Immunotherapy
Differentiation
Stem Cells Differentiated
cardiomyocytes
Stem cell
therapies
Aerobic Glycolysis Required for Optimal IFN-γ Production
Cell 153, 1239–1251, June 6, 2013 ª2013
Elsevier Inc. Erika Pearce et. al
For Research Use Only. Not for diagnostic purposes
The synergy of using Mass Spec with XF technology
0
50
100
150
200
250
300
350
400
Cumulative XF + MS Publication Count thru End
2015 ~2400
Total number of publications citing
XF data ~ 400
publications citing XF data and
Mass Spec data
For Research Use Only. Not for diagnostic purposes
Seahorse Cellular Metabolism and Agilent Molecular Metabolism
Video of inputs
and outputs
No zoom
Snap shots,
Fully zoomed
10,000x
Cellular Metabolism
Live cell rates
Molecular Metabolism
Pathway roadmaps
For Research Use Only. Not for diagnostic purposes
Multiplex iTRAQ Proteome Analysis
Caro, P. et. Al. Cancer cell. 16 Oct. 2012. 22(4) 547-560.
Oxygen
Consu
mption R
ate
(O
CR
)
Basal O
CR
(pm
ole
s/m
in)
Extracellular Flux (XF) analysis
MS+XF = Better Together!
DLBCL subsets
Oxidative subsets in DLBCL
For Research Use Only. Not for diagnostic purposes
Seahorse closes the loop in validation proteomic and genomic data Peroccchi et al1 leveraged data from comparative physiology, including OXPHOS studies, evolutionary genomics, organelle proteomics, and RNA interference studies to identify a product of CBARA1 gene as the founding member of a set of proteins required for high-capacity mitochondrial calcium uptake. Named MICU1 by the investigators, this gene product is associated with the mitochondrial inner membrane, and has several characteristics required for Ca2+ transport, including the two canonical EF hands essential for activity.
The investigators concluded that these
data show that OXPHOS remains intact in
MICU1-silenced cells, and that
mitochondrial Ca2+ uptake phenotype
does not occur as a secondary
consequence of generalized mitochondrial
dysfunction.
Dr. Nika Danial. Department of
Cell Biology Dana Farber
Cancer Institute
XF assay narrows search
Norberg E & Caro P et al. Caner Cell.
2012 Oct 16;22(4):547-60.
Targeted 13C flux provides answer
U13C-Palmitate
For Research Use Only. Not for diagnostic purposes.
XF Analyzers Measure the Two Major Energy Pathways of the Cell
Mitochondrial Respiration
……………… Measured by the
Oxygen Consumption Rate
OCR
Glycolysis ………………
Measured by the Extracellular
Acidification Rate
ECAR
For Research Use Only. Not for diagnostic purposes
For Research Use Only. Not for diagnostic purposes.
The transient micro-chamber enables rapid measurement of mitochondrial respiration and glycolysis in minutes
Well 1 Well 2
The bio-cartridge is
raised, bringing the system back
to baseline
The sensor cartridge is
raised, bringing the system
back to baseline
A transient micro-chamber
is formed
Measuring Cellular Bioenergetic Processes in Real Time
For Research Use Only. Not for diagnostic purposes.
Drug injection ports – the experimental possibilities are inifinite!
•4 drug injection ports per well
•Add inhibitors, stimulants, substrates,
and compounds
• Injections defined by the user [ NOTE: Video Requires QuickTime ]
For Research Use Only. Not for diagnostic purposes.
XFe Analyzers, FluxPaks, Stress Test Kits & Reagents
XF Stress Test Kits, XF phenotype
kit and reagents (PMP, BSA-PAL) XFe96, XFe24 and XFp
FluxPaks
XFe96 Analyzer XFe24 Analyzer XFp Analyzer
Mitochondrial Respiration
Oxidation of:
• Glucose
• Fatty Acids
• Amino Acids
Which Engine
(Pathway)?
Which Fuel
Source?
What questions can I answer?
Glycolysis
Fermentation of Glucose
For Research Use Only. Not for diagnostic purposes
Profiling Metabolic Engines: The XF Cell Energy Phenotype Test
Glycolysis
Mitochondria
l R
espiration
Aerobic
Glycolytic
Energetic
Oxyg
en
Co
nsu
mp
tio
n R
ate
(O
CR
)
Extracellular Acidification Rate (ECAR)
XF Energy Map
Memory T Cells
Activated T cells
Baseline
Neurons
Lymphoma Cells
Stressed
Neurons
Quiescent
Pairwise comparisons
Same? Different?
Comprehensive understanding of
your metabolic phenotype(s)
Baseline metabolisms
Stressed metabolisms
Metabolic Potential
For Research Use Only. Not for diagnostic purposes
XF Assays Measure Metabolic Switching
+Glc
-Glc
Substrate Induced Signaling Pathway Induced Gene Induced
0 0 40 60 20 80
100
300
200
400
OC
R (
pm
ol m
in-1
)
ECAR (mpH per min)
Fh1fl/fl
Fh1-/-CL1
Fh1-/-CL19
Hedgehog partial agonism drives Warburg-like
metabolism in muscle and brown fat.
Teperino R, et al., Cell. 2012 Oct 12;151(2):414-26.
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.
Haem oxygenase is synthetically lethal with the tumour
suppressor fumarate hydratase.
Frezza et al. Nature. 2011 Aug 17;477(7363):225-8.
Adipocytes +/- Hedgehog ligand Mantle cell lymphoma cells +/- glucose Kidney epithelial cells +/- fumarate hydratase
Aerobic More
Metabolic
Less
Metabolic Glycolytic
For Research Use Only. Not for diagnostic purposes
Cell Energy Phenotype Test
Analyzing metabolic functionality – Broad to Specific
Aerobic? Glycolytic?
Mito Stress Test
FAO
Glycolysis Stress
Test
Substrates
Isolated Mitochondria
Substrates
For Research Use Only. Not for diagnostic purposes
For Research Use Only. Not for diagnostic purposes.
van der Windt, JW, … and Pearce, EL, Washington School of Medicine. Immunity 2011 December 28
Naïve T cells
Effector T cells
Memory T cells
Mouse Lymphocytes
BASELINE 1 2 3
Mouse CD-1 Synaptosomes
Why the Mitochondrial Stress Test?
.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 1 2 3
Mouse CD-1 Synaptosomes
For Research Use Only. Not for diagnostic purposes.
XF Cell Mito Stress Test Profile
Measure the key parameters
of mitochondrial function:
• Basal Respiration
• ATP Production
• Maximal Respiration
and Spare Capacity
• Proton Leak
Basal
Respiration
ATP
Production
Proton Leak
Spare
Capacity
Maximal
Respiration
Non-mitochondrial Respiration
• Non-mitochondrial Respiration
XF Cell Mito Stress Test
For Research Use Only. Not for diagnostic purposes.
Non-mitochondrial
Basal
Resp.
ATP
turnover
Maximal
Respiration
H+ Leak
Oligomycin FCCP Rotenone +
Antimycin A
C2C12
The Mitochondrial Stress Test
I II III IV
V
H+
H+ H+ H+
O2 H20
ADP ATP
X
X X
Assessing electron flow through the ETC
OC
R (
pM
ole
s/m
in)
FCCP
Pyruvate
Malate
BASELINE 1 2 3
Mouse CD-1 Synaptosomes
For Research Use Only. Not for diagnostic purposes
For Research Use Only. Not for diagnostic purposes.
XF Glycolysis Stress Test Profile
Glycolytic
Capacity
Non-glycolytic Acidification
Glycolytic
Reserve
Capacity
Glycolysis Glycolysis
EC
AR
(m
pH
/min
)
Extr
acell
ula
r A
cid
ific
ati
on
E
CA
R (
mpH
/min
)
10mM
XF Glycolysis Stress Test
For Research Use Only. Not for diagnostic purposes.
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
Why the Glycolysis Stress Test?
XF Mito Fuel Flex Test
Glucose Glutamine Fatty Acids
nucleus
mitochondria
O2
Physiological
For Research Use Only. Not for diagnostic purposes
First demonstration of Seahorse to detect T cell activation opens up early activation window
July 11, 2017
Real time analysis of T cell activation
30
Gubser 2013 Nat Immunol
For Research Use Only. Not for diagnostic purposes
You don’t just get a more sensitive and faster assay, there’s more!
July 11, 2017
Real time analysis of T cell activation
31
Metabolic Requirements
Exogenous glucose
Pathway Requirements
mTOR
Signaling Requirements
CD28
Gubser, PM et al. 2013. Rapid effector function of memory CD8 T cells
requires an immediate-early glycolytic switch. Nature 14(10):1064-72.
For Research Use Only. Not for diagnostic purposes.
Glycolytic PER = Total PPR – CO2-dependent PPR
ECAR (mpH/min) * BF (mmol/L) * Vol ch (µL)
mit OCR (pmol/min)* CF
(pmol/min)
* Kvol
Improving proton flux as a measure of glycolysis
Functional metabolic data compliments your current data set
Mass Spectometry
End-point Assays
Flow Cytometry
qPCR
Only one company can deliver on both cellular and molecular metabolism – The unique differentiator
Cellular Metabolism
Molecular Metabolism
For Research Use Only. Not for diagnostic purposes
Questions?
Manager Seahorse XF Instruments
Midwest & West Coast
312.519.4352
For Research Use Only. Not for diagnostic purposes