April 18, 2017

Confidentiality Label

1

Dr. Daniel GebhardProduct Specialist (Seahorse Consumables)

Baseline

Stressed

Glycolytic

Aerobic Energetic

Quiescent

2For Research Use Only. Not for use in diagnostic procedures

Seahorse is the most used, and mostcited measure of functional metabolism

>1,500 instruments >10,000 users

>2,000 publications700

750

2015Projected

No matter what you call it … metabolism affects every therapeutic area

Hegedus et al

W. Van der Windt et al

LeBleu et al

Yin et al

Wang et al

Bettum et al

Ma et al

April 18, 2017

Confidentiality Label

4For Research Use Only. Not for use in diagnostic procedures

XF Power-Pak

A Seahorse for every application and every lab

XFe96 Analyzer

New!

XFp Analyzer XFe24 Analyzer

April 18, 2017

Confidentiality Label

5For Research Use Only. Not for use in diagnostic procedures

Seahorse can answer your metabolic questions

Mitochondrial function

Glycolytic function

Metabolic phenotype

Glucose

Glutamine

Fatty acids

ENGINES

FUELS

April 18, 2017

Confidentiality Label

6For Research Use Only. Not for use in diagnostic procedures

Scientists Want to Know the Answers to These Questions

MitochondrialRespirationOxidation of:• Glucose• Fatty Acids• Amino Acids

Which Pathway?

Fuel Source?

Metabolism: Engines and Fuels

GlycolysisFermentation ofGlucose

Synergy between XF and MS

Video of inputs and outputs

No zoom

Snap shots, Fully zoomed10,000x

Cellular MetabolismLive cell rates

Molecular MetabolismPathway roadmaps

For Research Use Only. Not for use in diagnostic procedures

April 18, 2017

Confidentiality Label

8For Research Use Only. Not for use in diagnostic procedures

XFe96 Cell Culture Microplate

XF Assay Medium

Solid State Sensor

Cell Monolayer

Sensor Probes

Fiber Optics

Well 1 Well 2

Measuring Mitochondrial Respiration and Glycolysis in a Microplate

April 18, 2017Confidentiality Label

9For Research Use Only. Not for use in diagnostic procedures

A temporary 2.28 µl micro-

chamber is formed

The bio-cartridge is

raised, bringing the system back to baseline

The bio-cartridge is

raised, bringing the system back to baseline

The cartridge is raised,

bringing the system back to baseline

Measuring Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR)

XFe96 Cell Culture Microplate

XF Assay Medium

Solid State Sensor

Cell Monolayer

Sensor Probes

April 18, 2017

10For Research Use Only. Not for use in diagnostic procedures

The slopes of level decreases are converted into ratesO

2 (m

mH

g)O

CR

(pm

ol/m

in)

April 18, 2017

Confidentiality Label

11For Research Use Only. Not for use in diagnostic procedures

Automated Drug Injection Ports Enable Kinetic, Functional Data

• 4 drug injection ports per well

• Add inhibitors, stimulants, substrates, and compounds

• Injections defined by the user

Injects up to 4 compounds

[ NOTE: Video Requires QuickTime ]

April 18, 2017

Confidentiality Label

12For Research Use Only. Not for use in diagnostic procedures

XF Assay Flow Chart

Prepare cells in XF plate

Seed cells and incubate overnight in growth medium

Change medium to bicarbonate-free low-

buffered assay medium

Prepare sensor cartridge

Hydrate sensor cartridge overnight

Calibrate sensors

Assay

Add compounds to reagent ports

April 18, 2017

Confidentiality Label

13For Research Use Only. Not for use in diagnostic procedures

Software: Wave 2.3!

April 18, 2017

Confidentiality Label

14For Research Use Only. Not for use in diagnostic procedures

XF Cell Energy Phenotype Test

Seahorse Gold Standard assays are the key metabolic assays

XF Mito Fuel Flex Test

Glucose Pathway

Flexibility

Dependency

Capacity

100

80

60

40

20

0Glutamine Pathway

80

60

40

20

0

Flexibility

Dependency

Capacity

Fatty Acid Pathway

Flexibility

80

60

40

20

0

Dependency

Capacity

Flexibility

Glu

cose

Oxi

dat

ion

(% o

f G

LC+G

LN+F

A O

xid

atio

n)

Glu

tam

ine

Oxi

dat

ion

(% o

f G

LC+G

LN+F

A O

xid

atio

n)

Fatt

y A

cid

Oxi

dat

ion

(% o

f G

LC+G

LN+F

A O

xid

atio

n)

100 100

XF Cell Mito Stress Test XF Glycolysis Stress Test

April 18, 2017

Confidentiality Label

15For Research Use Only. Not for use in diagnostic procedures

Metabolic Engines: XF Energy Mapand XF Cell Energy Phenotype Test

• Define XF Metabolic Phenotypes

Glycolysis

Mit

och

on

dri

al R

esp

irat

ion

Aerobic

Glycolytic

Energetic

Oxy

gen

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-/-CL1Kidney Cells

• Monitor Cell Quality and Health

Quiescent

April 18, 2017

Confidentiality Label

16For Research Use Only. Not for use in diagnostic procedures

• Maximal Respiration and Spare Capacity

BasalRespiration

ATPProduction

Proton Leak

XF Cell Mito Stress Test

XF Cell Mito Stress Test Profile

Spare Capacity

MaximalRespiration

Non-mitochondrial Respiration

Measure the key parameters of mitochondrial function:• Basal Respiration

• ATP Production

• Proton Leak

Mitochondrial spare capacity enables cells to respond to acute stress

April 18, 2017

Confidentiality Label

17For Research Use Only. Not for use in diagnostic procedures

Mito Stress Test combined with substrate or inhibitorsFatty Acid Oxidation Profile Example

Seahorse XF Fatty Acid Oxidation Profile

Making cells reliant on exogenous palmitate in order to investigate effects of KLF15 and PPARα on FAO

Domenick A. Prosdocimo, Jenine E. John, LileiZhang, et al., “KLF15 and PPARα Cooperate to Regulate Cardiomyocyte Lipid Gene Expression and Oxidation,” PPAR Research, vol. 2015, Article ID 201625, 2015. doi:10.1155/2015/201625

+/- KLF15 sh-RNA

WY = WY 14643 PPARα agonist

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 zoomed10,000x

Cellular MetabolismLive cell rates

Molecular MetabolismPathway roadmaps

For Research Use Only. Not for use in diagnostic procedures

DLBCL OxPhos cell line panelDBLC non-OxPhos cell line panel

XF assays target and guide metabolomic/flux studies

Metabolic Signatures Uncover Distinct Targets in MolecularSubsets of Diffuse Large B-Cell LymphomaPilar Caro et. al. Cancer Cell. 2012 October 16; 22(4):

For Research Use Only. Not for use in diagnostic procedures

Metabolomics and 13C flux data enhanced and extended by Seahorse XF assays

Metabolomics narrowed search to a gene product that delivers pyruvate to the mitochondria

✕Pyruvate

Seahorse provided function confirmation and clinical relevance

WT KO WT KO

Jared Rutter PhD, University of Utah

A Mitochondrial Pyruvate Carrier Required for Pyruvate Uptakein Yeast, Drosophila, and Humans Bricker DK, et al. Science. 2012 Jul 6;337(6090):96-100

For Research Use Only. Not for use in diagnostic procedures

Tumor growth requires reductive glutamine metabolism to maintain redox homeostasis

13C glutamine

Cytosolic reductive carboxylation is required for mitochondrial redox homeostasis during anchorage-independent cell growthJiang L, et al, Nature. 2016 Apr14;532(7598):255-8

For Research Use Only. Not for use in diagnostic procedures

Precise measurements of oxygen consumption were required to complete the analysis

Cytosolic reductive carboxylation is required for mitochondrial redox homeostasis during anchorage-independent cell growthJiang L, et al, Nature. 2016 Apr14;532(7598):255-8

For Research Use Only. Not for use in diagnostic procedures

April 18, 2017

Confidentiality Label

24For Research Use Only. Not for use in diagnostic procedures

Questions?

THANK YOU!