Download Research

Gene-Protein-metabolism links
Cell Metabolism Assays
for
OMICS
Research
STANDARD Parameters of
Functional METABOLIsm
Genomics
Cells use gene expression to synthesize proteins and other products that are essential for various cellular
functions. Gene expression levels, while associated with metabolism, cannot solely predict the functional impact on
metabolism. This creates a gap in understanding the complex physiological relation between genes and metabolism.
Researchers are using XF Technology to link functional metabolism to gene expression for a complete picture. The
XF Cell Mito Stress Test measures the key parameters of mitochondrial respiration: basal respiration, ATP-linked
respiration, maximal respiration, and spare respiratory capacity. The XF Glycolysis Stress Test measures the key
parameters of glycolytic activity: glycolysis, glycolytic capacity, glycolytic reserve.
Oxygen Consumption Rate (OCR)
OCR
(pmol/min)
(pmol/min)
500
Control
Hdac9 deleted
*
400
300
200
100
FCCP
FCCP
Mouse T cells
pmol O2 per min per µg protein
Oligomycin
oligomycin
Basal
basal
Basal respiration
4
pmol O2 per min per mg protein
0
P<0.05
3
2
XF Assay reveals a link between histone deacetylase 9 (Hdac9) deletion and mitochondrial
1
respiration correlating to a genetic and functional increase in T regulatory cells.
(Beier UH et al., 2015. FASEB J.)
0
L
P<0.05
4
2
0
1
iPS
S-
LA
ME
3
iPS
S-
LA
ME
2
iPS
S-
LA
ME
iPS
S-
LA
ME
Maximum respiration
6
iPS
S-
iPS
Spare
6 Capacity
P<0.05
Oxidative
reserve
3
4
2
2
1
0
1
4 P<0.05
2
0
P<0.05
3
2
1
3
2
iPS -iPS
iPS -iPS
iPS
SSS
S
SLA ELA
LA LA
LA
ME ME
M
ME
1
S1
S33
S22
-iPS
-iPS
-iPS
SiP
A-SiP
A-SiP
LS
LSAELS
M
M
M
LEA
LA
LEA
ME
S1
-iP
S
LA
ME
iPS
S-
LA
ME
ME
ME
1
0
3 S1
S2 PSS33
SS22
S1
iPS i-P
-i
S- AS
-SiP-iP
- iP LAS-iPSA-SiP
LA ALS
LSA
A
E
LEAL
L
ME EMLE
ME EM
EM
M
MELAS-iPS1
2
1
0
10
5
1
iPS
S-
LA
ME
15
MELAS-iPS2
2
1
S3 S1 iPS2 PS3
i
-iP S-iP
SAS
AS
0 MEL ELA MEL ELA
M
M
0
25
P<0.05
4
2
0
S11
-iPS
SiP
LSA-
ME
3
22
PS3
PS
S
iP-iS
iSP-iS
LAS
SLAE
E
A
A
LM
LM
OCT4
SOX1
PAX6
10000
1000
NANOG
NESTIN
100
10
1
0.1
0.01
0.001
MELAS-IPS1 MELAS-IPS2 MELAS-IPS3
2
50
75
iPS
S-
LA
ME
P<0.05
2
1
0
100
Human neuronal progenitor cells
XF Cell Mito Stress Test predicts differentiation status in neuronal progenitor cells (NPCs) derived from patients with mutations
causing mitochondrial encepharopathy and stroke-like episodes (MELAS) which correlates to gene expression.
S1
-iP
AS
ME
100000
3
iPS
S-
LA
ME
3
L
ME
(min)
TimeTime
(min)
(Ma H et al., 2015. Nature.)
1
iPS
S-
Oxidative reserv
iPS
S-
LA
ME
0
LA
MELAS-iPS3
&
MELAS-iPS1
MELAS-iPS2 Rotenone
MELAS-iPS3
3 Oligomycin FCCP
antimycin
RotenoneA&
P<0.05
P<0.05
antimycin A
Oligomycin
FCCP
0
6
E
M
LA
ME
M
Oxidative reserve
Oxidative reserve
3
1
ME
pmol O2 per min per µg protein
P<0.05
2
M
Maximum respiration
Maximum respiration
6
E
M0
0
S-
LA
ME
ME
1
S2
-iP
S
LA
2
L
ME
2
iPS
AS
2
Maximal Respiration
Oxygen Consumption Rate (OCR)
(pmol/min/µg
protein)
pmol
O2 per min per
µg protein
1
Oxygen Consumption Rate (OCR)
µg protein
pmol
O2 per min per protein)
(pmol/min/µg
2
LA
ME
pmol O2 per min per µg protein
1
0
P<0.05
3 P<0.05
3 P<0.05
L
ME
3
iPS
AS
P<0.05
Maximum respiration
Relative Expression Levels
iP
S-
LA
ME
S2
1
3
pmol O2 per min per µg protein
iP
S-
LA
ME
S3
2
2
pmol O2 per min per µg protein
iP
S-
LA
ME
S1
P<0.05
ATP turnover
33
0
SiP3S3 PS1 -SiP2S2 -iPS3
SS11
-AiP
-iSP-iP
S- AS-i S-AiP
S
S
A
S
S
0EL
AEL MEL LMAEL MELA
A
L
L
M
M
1
2
ME
M3E
ME
Oxygen Consumption Rate (OCR)
per min
per
µg protein
pmol
O2pmol
O2 per
min
per µg protein
(pmol/min/µg
protein)
0
4
pmol O2 per min per µg protein
1
4
ATP Production
ATP turnover
ATP turnover
OxygenOxygen
Consumption
Rate
(OCR)
Consumption
Rate
(pmol/min/µg
protein)
(pmol O2 per µgprotein)
2
Basal respiration
Basal respiration
pmol O2 per min per mg protein
P<0.05
3
Basal Respiration
per
min
permin
µg protein
pmol O2pmol
(pmol/min/mg
protein)
O2 per
per mg
protein
Basal respiration
4
Oxygen Consumption Rate (OCR)
ME
1
iPS
AS
ATP turnover
3
S3
-iP
AS
L
ME
iPS
S-
LA
ME
THE WORLD’S MOST ADVANCED
METABOLIC ANALYZERS
Proven Technology for Cutting Edge Research
There are over 2,000 references utilizing XF Technology published
in leading journals such as Nature and Cell. Scientists are
embracing XF Technology to identify metabolic phenotypes and
reprogramming to target metabolic pathways for therapeutic
purposes.
Glucose
Pyruvate
Lactate
IV
III
II
GLYCOLYSIS —
ECAR
(Extracellular
Acidification Rate)
MITOCHONDRIAL
RESPIRATION —
OCR (Oxygen
Consumption Rate)
I
Cells generate ATP
Mitochondria consume
via glycolysis independent
oxygen when oxidizing fatty
of oxygen, producing lactic acid
acids or other substrates
and protons. XF Analyzers measure
to generate ATP. XF
glycolysis by measuring the extracel-
Analyzers measure mitochondrial
lular acidification rate (ECAR) of
respiration by measuring the oxygen
cells.
consumption rate (OCR) of cells.
Proteomics
Advances in proteomic technology enables genome-wide data analysis, pinpointing specific proteins and expression
levels. Proteomic data directly links to metabolism without requiring the regulatory mechanisms associated with mRNA
expression and final protein expression. However, conclusions utilizing only these types of experiments are limited.
XF Technology not only confirms proteomic data but also allows for further investigation of protein function.
WT
PKA-deficient
2.0 +
1.50-1.99
0.51-0.70
0.5-
ATP-linked Respiration Rate
(pmol O2 /min/ 1x105 cells)
150
*
50
0
CPT-cAMP
-
+
-
+
Murine lymphoma cells
M-BMM
M-BMM
M-BMM
GM-BMM
Glucose Oligomycin 2-DG
Glucose
Oligomycin 2-DG
0.71-1.49
Not represented
100
GM-BMM
GM-BMM
GM-BMM
Microarray Fold Change
50
Extracellular Acidification Rate (ECAR)
(mpH/min)
200
Glucose Oligomycin
50
40
GO_BP:
Gene & Protein Expression Profile
M-BMM
Glucose metabolic process
mRNA
mRNA
2-DG
RPIA
PYGL
PFKP
LDHB
GYS1
FABP5
ENO1
ALDOC
40
30
50
3020
10
20
40
10
0
30
0
20
***
1
1
30
30
10
0
60
0
0 0
0
90 120 GM-BMM
15 18 21 24
Time Glucose
(min)Oligomycin
60
***
M-BMM
2-DG
9500 120 150 180 210 240
40
Time
(min)
30
20
***
10
1
30
0
610
30
0
protein
protein
1
2
3 10
relative expression level
GM-BMM/ M-BMM
12
***
9600 90121200 1510 51080 2110 82040 210 240
Time (min)
Time (min)
Murine bone-derived macrophages
XF Cell Mito Stress Test reveals that protein kinase A (PKA)dependent changes in protein and gene expression
correlate with changes in ATP-linked respiration.
XF Glycolysis Stress Test reveals the increased glycolytic capacity
of granulocyte-stimulated macrophages (M-BMM), confirming the
inherently higher glycolytic protein expression.
(Wilderman A et al., 2015. J Biol Chem.)
(Na YR et al., 2015. Mol Cell Proteomics.)
MEASURING THE KEY PARAMETERS
OF Functional Cell metabolism
Metabolomics
Techniques, such as mass spectrometry (MS) and nuclear magnetic resonance
(NMR), enable the analysis of hundreds of metabolites in a given sample.
However, the data from these techniques alone are not sufficient to assess
functionality within a biological context. XF Technology provides real-time analysis
of cellular response to stress or nutrients, and can direct metabolomics research,
adding efficiency and direction to any area of research.
**
0
F6P
FBP
1 2
pmol/106 cells
350
3
G3P
* **
350
0
3PG
1
2
3
0
1
2
PEP
3
20,000
pmol/106 cells
pmol/106 cells
**
175
175
ATP
60
R5P
X5P
E4P
*
10,000
0
1 2 3
50,000
25,000
0
Lactate
**
LDHs
PKMs
Pyruvate
PDHs
**
0
1
2
3
2
3
*
90
45
0
300
1
“With very little learning curve,
XF Technology provides highquality data. We generated
publishable results within the
first month.”
***
***
150
0
1
120
**
60
2
3
**
0
1
2
1,000
*
3
500
0
Acetyl-CoA
1 2 3
Medical School
40
3
*
1,250
Ru5P
6PG
*
80
pmol/106 cells
pmol/106 cells
2
G6P
1 2 3
pmol/106 cells
1
2,500
10
pmol/106 cells
0
100
pmol/106 cells
175
*
***
1,000
pmol/106 cells
GLUTs
HKs
1 2 3
- Researcher at Case Western
PC9
GEF
ERLO
DMSO
GEF
ERLO
ERLO
DMSO
0
GEF
Reserve University
HCC827
H1975
Human lung adenocarcinoma cells
XF Glycolysis Stress Test predicts sensitivity to first-line EGFR inhibitors used in lung
adenocarcinoma cells, and correlates to reduced metabolite levels.
Extracellular
AcidificationRate
Rate
(ECAR)
Extracelluar
(ECAR)
Extracelluar Acidification
Acidification
Rate
(ECAR)
(mpH/min)
Extracelluar (mpH/min)
Acidification
Rate (ECAR)
(mpH/min)
(Makinoshima H et al., 2014. J Biol Chem.)
Th1
Th1
Th1
Th17
Th17
Th17
Th17
Th1
60
60
Treg
Treg
Treg
Treg
Glucose
Glucose
Oligomycin
Oligomycin
Glucose
Oligomycin
2-DG
California, San Diego
50
(mpH/min)
40
40
40
30
30
30
1
20
20
Oxygen
Consumption
Rate (OCR)
Oxygen
Consumption
Rate
Oxygen
Consumption
Rate (OCR)
(OCR)
Oxygen Consumption Rate (OCR)
(pmol/min)
(pmol/min)
(pmol/min)
(pmol/min)
0
G6P/F6P
Oxidative PPP
Th1
Th17
Treg
6P
Gluconate
15
10
00
8
6
4
2
0
1.5
Glucose 0.5
Glycolysis
20
10
5
Relative level
normalized to naive
(red line)
0
20
15
0
00
1400
1400
1400
25
50
25
25
75
Time (min)
50
50
100
75
75
125
150
100
100 125
125
Rotenone &
Oligomycin FCCP antimycin
Rotenone
Rotenone
&&
A
Fructose 10
1,6-P 5
20
15
150
150
Glycerol
3P
5
0
0
2.5
2
1.5
1
0.5
0
0
5
4
3
0
1
5
4
3
2
1
0
Lactate
PEP 0.5
0
3
2
Pyruvate 1
0
Sedoheptulose
7P
Eryhrose
4P
1
1,3-P
Glycerate 0.5
1.5
600
800
800
5
1.5
Glycerate 2
1
3P
25
20
15
10
5
0
15
10
G3P
DHAP 10
1200
1000
1000
800
Nonoxidative PPP
0
5
4
3
2
1
0
Oligomycin
Oligomycin FCCP
FCCP antimycin
antimycinAA
1200
1200
1000
2
1.5
1
0.5
0
CisAconitate
Citrate
2
1.5
1
0.5
0
5
4
3
2
1
0
6
2.5
2
1.5
1
0.5
0
Succinate
4
2
6
0
Oxaloacetate 4
Acetyl CoA
α-Keto
glutarate
TCA cycle
Fumarate
2
0
Malate
400
600
600
200
400
400
0
0
200
200
00
00
25
50
75
Time (min)
Time
(min)
100
125
Murine T cells
25
25
50
50
75
75
Time
Time(min)
(min)
“When we decided to more
closely investigate branched chain amino acid metabolism,
we chose to incorporate our
XF Technology, knowing that it
is a powerful tool for assessing
mitochondrial function and
metabolism.”
- Researcher at University of
2-DG
2-DG
60
50
50
10
100
“We were really surprised by the
impact of measuring metabolism
in our model system. This opens
up new research avenues for us.”
- Researcher at Rosalind Franklin
10,000
pmol/106 cells
pmol/106 cells
Glucose
350
DHAP
DMSO
Extracellular Acidification Rate (ECAR)
ECAR
(mpH/min)
(mpH/min)
ERLO
**
***
***
***
120
GEF
pmol/106 cells
GEF=
Gefitinib
DMSO
ERLO= Erolotinib
What OUR Customers
ARE SAYING
150
100
100 125
125 150
150
XF Glycolysis Stress Test and XF Cell Mito Stress Test reveal increased levels of mitochondrial
respiration and glycolytic activity in TH17 (T cells) which correlate to changes in metabolite levels.
(Gerriets VA et al., 2015. J Clin Invest.)
Functional metabolic assayS
The Genotype-Phenotype Link
The traditional paradigm encompassing the flow of information from gene to protein to function can be found
in nearly every biology and biochemistry textbook. At the time, this discovery was thought to explain all of biology,
health, and disease. However, only focusing on the expression of a single gene or protein neglects the multi-faceted
consequences of that expression on metabolism, as well as overall cell and tissue health.
Mitochondria are at the crux of cellular energy generation, intracellular
signaling, and cell death and survival regulation. Compromised
mitochondria can strain the expansive and intricate cellular metabolic
network. There is increasing appreciation for the role of mitochondria
in disease pathology, etiology, and cell health.
Integrating processes that demonstrate the link amongst genes,
proteins, and metabolism allows researchers to analytically answer
their scientific questions. XF Technology provides researchers the
necessary tools to measure the functional, metabolic impact of gene
and protein expression, faster and easier using live cells.
GENES & METABOLISM LINK
Gene
Expression
Functional
Metabolism
Full
Picture
of Cell
Health
Protein
Expression
substrates and metabolism
GLUCOSE
GLUCOSE
PI3/AKT
p53
GLYCOLYSIS
PYRUVATE
Acetyl-CoA
TCA
LIPID SYNTHESIS
GLUTAMATE
GLUTAMATE
PI3/AKT
ETC
TCA
GLYCOLYSIS
(ECAR)
MYC
GLUTAMINOLYSIS
CITRATE
PYRUVATE
HIF1
MYC
FATTY ACID
LACTATE
MYC
HIF1
HIF1
FATTY ACID
MITOCHONDRIAL
RESPIRATION
(OCR)
GLUTAMINE
Gold Standard metabolic Assays
MEASURING THE KEY PARAMETERS OF CELL METABOLISM
XF Glycolysis Stress Test Profile
XF Cell Mito Stress Test Profile
Glycolytic Function
Mitochondrial Respiration
FCCP
Extracellular Acidification Rate (ECAR)
(mpH/min)
Oxygen Consumption Rate (OCR)
(pmol/min)
Oligomycin
Rotenone &
antimycin A
360
320
280
Spare
Capacity
240
200
160
120
80
Basal
Respiration
Maximal
Respiration
Proton Leak
Non-mitochondrial Oxygen Consumption
40
0
ATP
Production
0
10
20 30 40 50 60 70
Time (minutes)
80 90 100 110
Glucose
Oligomycin
2-DG
45
40
35
Glycolytic
Reserve
30
25
Glycolytic
Capacity
20
15
Glycolysis
10
5
0
Non-glycolytic Acidification
0
10
20
30
40 50 60 70
Time (minutes)
80
90 100
XF Mito Fuel Flex Test Profile
XF Cell Energy Phenotype Test
XF Mito Fuel Flex Test Profile
Metabolic
Phenotype
& Potential
XFp
Cell Energy
Phenotype
Profile
Mitochondrial
MitochondrialFunction
Function
Baseline
Phenotype
Quiescent
Glycolytic
Glycolysis
Extracellular Acidification
Acidification Rate
(ECAR)
Extracellular
Rate
(ECAR)
60
Flexibility
40
Flexibility
20
Dependency
0
Glucose Pathway
Glucose Pathway
Dependency
Dependency
Glutamine Pathway Fatty Acid Pathway
Glutamine Pathway Fatty Acid Pathway
Corporate Headquarters
European Headquarters
Asia-Pacific Headquarters
Seahorse Bioscience Inc.
16 Esquire Road
North Billerica, MA 01862 US
Phone: +1.978.671.1600
www.seahorsebio.com
Seahorse Bioscience Europe
Fruebjergvej 3
2100 Copenhagen DK
Phone: +45 31 36 98 78
Seahorse Bioscience Asia
199 Guo Shou Jing Rd, Suite 207
Pudong, Shanghai 201203 CN
Phone: 0086 21 33901768
© 2015 All rights reserved. Seahorse Bioscience and the Seahorse logo are trademarks of Seahorse Bioscience Inc.
The content in this brochure is for informational purposes only and may be subject to change without notice.
Rev 1
n
Capacity
te
Capacity
ab
Po
80
Capacity
M
et
ic
ol
l
tia
% GLC+GLN+FA
% GLC+GLN+FA
FuelOxidation
Oxidation
Stressed
Phenotype
Mitochondrial Respiration
OxygenConsumption
Consumption Rate
Oxygen
Rate(OCR)
(OCR)
100
Energetic
Aerobic