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Measuring Mitochondrial function and Glycolysis for the Metabolic Phenotype detection in Hematological Diseases
Emma Nora Di Capua, PhD
Seahorse Product Specialist
Italy/Spain
October 4, 2017
Confidentiality Label
1
For Research Use Only. Not for use in diagnostic procedures.
Cells are not static, they are dynamicMany tools analyze at a single time-point, few measure in real-time
October 4, 2017
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2
t=0
t=1
t=2
StaticDynamic
For Research Use Only. Not for use in diagnostic procedures.
Seahorse XF assays go beyond what cells are to reveal what they do
October 4, 2017
Confidentiality Label
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Proliferation Activation
Differentiation Death
For Research Use Only. Not for use in diagnostic procedures.
Seahorse XF assays go beyond what cells are to reveal what they do
October 4, 2017
Confidentiality Label
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Follow immune cell differentiation
Real time activation
Infection Effector expansion Memory
Modulate metabolism to control outcomes
Naïve CD4+
T cell
Th1
Th2
Th17
Treg
Tfh
Determine cell state-specific metabolic dependency
For Research Use Only. Not for use in diagnostic procedures.
XF technology empowers you to answer your questions about cellular function in real-time
October 4, 2017
Confidentiality Label
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Real-time kinetic
measurements
Integrated
injection
ports for real-
time compound
testing
Label-free
multiparameter
sensorH+ & O2
Ready-to-
use
validated
kits &
reagents
Advanced
software &
analytics
Live cell
analysis with 2D & 3D
plate options
For Research Use Only. Not for use in diagnostic procedures.
Metabolic inputs are a prerequisite for all cell function
October 4, 2017
Confidentiality Label
6
XF assays measure the rates of change of
the two key energy metabolism pathways
Glycolysis
[H+]Proton effluxrate
Respiration
[Oxygen]Oxygen
consumptionrate
For Research Use Only. Not for use in diagnostic procedures.
Standardized assays assess metabolic phenotype, glycolytic and mitochondrial function and cellular fuel usage
October 4, 2017
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Glycolytic Function Mitochondrial Function
Metabolic Phenotype Fuel Usage and Liability
Real time analysis of Immune Cells
The gold standard assay to
interrogate mitochondrial
functionality
Profile cellular glycolysis.
Quantify glycolytic rate and
pathway capacity
Determine how energetic your cells
are, preferred energy pathway and
pathway switching in response to
stimulation
Understand cell fuel/substrate
metabolism from three fuel
sources. Identify preference,
flexibility and fuel liability
Any cell
4 compound
injection ports
Real-time, multi-parameter
kinetics
For Research Use Only. Not for use in diagnostic procedures.
Loss of mitochondrial carrier homologue 2 (MTCH2), increases mitochondrial OXPHOS, triggering HSC entry into cycle
October 4, 2017
Confidentiality Label
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Live cell analysis of oxygen
consumption rate enables
identification of a novel pathway
indispensable in maintaining
HSC homeostasis
Maryanovich 2015 Nature
For Research Use Only. Not for use in diagnostic procedures.
The different cell state-specific dependency inhematopoiesis and leukemogenesis as potential therapeuticopportunity
October 4, 2017
Confidentiality Label
9
Measuring OCR they confirm
the hypothesis that PKM2
deletion with PKM1 expression
impairs AG, enhances oxidative
metabolism and limits
macromolecule biosynthesis
required for cell proliferation.
Wang et al 2014 Cell
For Research Use Only. Not for use in diagnostic procedures.
Understand Cellular and Molecular Metabolism Agilent Seahorse and Mass Spectrometry Together
Cellular
Metabolism
Molecular
Metabolism
For Research Use Only. Not for use in diagnostic procedures.
Metabolism – a lot you can look at
Many don’t know where to start11
For Research Use Only. Not for use in diagnostic procedures.
But if something changed the cell’s
behavior…
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….Seahorse sees it! ...in real time!!
For Research Use Only. Not for use in diagnostic procedures.
For Research Use Only. Not for use in diagnostic procedures.
Synergy between XF and MS
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 use in diagnostic procedures.
Real time metabolic analysis delivers a rapid and robust indicator of activation in minutes
October 4, 2017
Confidentiality Label
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Use injection ports to
investigate modulators of T cell
activation kinetics and
magnitude.
www.agilent.com/chem/immunology
For Research Use Only. Not for use in diagnostic procedures.
Seahorse is the most rapid, user friendly way to measure energy metabolism
October 4, 2017
Confidentiality Label
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Four programmable
injection ports per well
O2 sensor to measure
mitochondrial respiration
H+ sensor to
measure glycolysis
Resuspend kit reagents
and prepare cells
Pair reagent-loaded sensor
cartridge with cell culture plate
Run assay on your
XF analyzer
For Research Use Only. Not for use in diagnostic procedures.
Seahorse XF assays go beyond what cells are to reveal what they are doing
October 4, 2017
Confidentiality Label
17
• Dynamic biologyRead-out functional changes as they occur
• Highly sensitiveNever miss an unexpected response.
• Complete pictureSimultaneously measure key metabolic pathways
• Data richAutomated compound injection enables real-time in situ modulation.
• More relevantMeasure any cells, mitochondria, spheroids, islets, more.
• EfficientRequires <40-200Kcells per well.
• Reduce ComplexityNo need to select appropriate labels, arrays, panels, etc.
• Non-invasiveWon’t interfere with cell function
Multi-parameter
Real-Time Kinetics
In situ Label-free
For Research Use Only. Not for use in diagnostic procedures. October 4, 2017
Confidentiality Label
18
What are our next steps?
We can work with you on a customized plan to explore XF Technology, with options including
• Webinars
• Cell Reference Database
• Website
Questions?
THANK YOU!Emma Nora Di Capua, PhD
0039 3346443983