O 2 O 2
O 2
O 2 O 2
O 2
+ H
+ H
+ H
+ H + H
H +
O 2
O 2
O 2
Brown Hao Product Specialist Cold Spring Biotech Inc.
冷泉港生物科技股份有限公司 E-mail: [email protected]
A Better Option: Real-Time Cell Metabolism in a Microplate
XFe Analyzers Measure the Two Major Energy Pathways
Extracellular acidification rate
(ECAR)
= Glycolysis
Oxygen Consumption Rate (OCR)
= Mitochondrial Respiration
XF Cell Culture Microplate
Assay Medium
Solid State Sensor
Cell Monolayer
Sensor Probes
Fiber Optics
Well 1 Well 2
Measuring Mitochondrial Respiration and Glycolysis in a Microplate
The transient micro-chamber enables rapid measurement of mitochondrial respiration and glycolysis in minutes
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
Well 1 Well 2
Measuring Mitochondrial Respiration and Glycolysis in a Microplate
Well 1 Well 2
Time (min)
100
120
130
140
150
160
1 2 3 4
7.20
7.25
7.30
7.35
7.40
7.45
pH (E
CAR)
5 6 7
mm
Hg
(OCR
)
C2 C2 C2 C2
C2
C2 C2
C2
C2
C2 C2
C2
C2
C2
C2
C2
C2
C2
C2 C2
C2
C2 C2
C2
C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2
C2 C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2 C2
C2
C2
C2
C2 C2 C2 C2 C2
A temporary microchamber 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 bio-cartridge is raised, bringing the system back to baseline ECAR
OCR
The rate is calculated from the slope
Metabolism in a Microplate- Cellular bioenergetics for the 21st century
Automated Drug Injection Ports Enable Kinetic, Functional Data
• 4 drug injection ports per well
• Add inhibitors, stimulants, substrates, and compounds
• Injections are defined by the user
What is making XF Analyzer unique for scientists
Platform to simultaneously measure two parameters (OCR and ECAR)
Rapid, real- time and label-free technology
Microplate format and high throughput
Wide kinds of cells: Assays adherent cells, suspension cells and
subcellular components
Up to four injection ports
Sensitive and precision optical microsensor detection system
Intuitive software with touch screen interface and data analysis system
Seahorse XF Extracellular Flux Analyzers, FluxPaks & Kits
XFe 24细胞代谢分析仪 XFe 96细胞代谢分析仪
XF测试样品制备仪
Application
神经退行性疾病
肥胖、糖尿病和代谢紊乱
线粒体疾病
衰老
癌症
转发医学
模式生物
毒性学 心血管疾病
免疫学
药物筛选
Krebs Cycle
e- e-
e- e-
H+H+ H+H+
Cyt C e- e-
H+H+ O2 H+H+ H2O
ADP ATP
Cyt C
e-
O2
Q
H+`H+
NADH
NAD+
H+ H+ H+ H+H+
H+
Cyt C
H+`
e-
e- e-
IN
OUT
Peter S. Rabinwitch after Mandavilli et al, Mutation Research 509 (2002) 127–151
Mitochondrial Electron Transport Chain
2OH- OH-
Oligomycin
FCCP
Antimycin A & Rotenone
Basal Respiration
ATP Production
Proton Leak
XF CELL MITO STRESS TEST The Gold Standard That Makes You a Bioenergetics Expert
XF Cell Mito Stress Test Profile
Spare Capacity
Maximal Respiration
Non-mitochondrial Respiration
Kit measures the key parameters of mitochondrial function: • Basal Respiration
• ATP Production
• Maximal and Spare Capacity
• Proton Leak
XF GLYCOLYSIS STRESS TEST XF Glycolysis Stress Test Kit
XF Glycolysis Stress Test Profile
Glycolytic Capacity
Non-glycolytic Acidification
Glycolytic Reserve Capacity
Measures the key parameters of glycolytic function:
• Non-glycolytic Acidification
• Glycolysis
• Glycolytic Capacity
• Glycolytic Reserve Glycolysis Glycolysis EC
AR
(mpH
/min
) Ex
trac
ellu
lar A
cidi
ficat
ion
ECA
R (m
pH/m
in)
10mM
XFe Metabolic Phenotype Assay - PhenoGram
Measures the metabolic state of cells:
Warburg Effect
Reverse Warburg Effect
Crabtree Effect
Pasteur Effect
• More Metabolic
• Less Metabolic
• Glycolytic
• Aerobic
Memory T Cells
Neurons
H460 Lung Cancer Cells
Pluripotent Stem Cells
XFe Assays Measure Metabolic Switching (Cancer, Diabetes and Obesity)
+Glc
-Glc
Substrate Induced Signaling Pathway Induced Gene Induced
0 0 40 60 20 80
100
300
200
400
OCR
(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
Seahorse Simplifies Measuring Cellular Metabolism
XF Cell Mito Stress Test Profile
TRANSLATIONAL MEDICINE Stress Test Identifies Mitochondrial Dysfunction in Diseases
Data courtesy of Dr. Pinar Coskun, Department of Neurobiology and Behavior at UC Irvine
Parkinson’s Disease Lower spare capacity in patient
HIV * Lower spare capacity in patients
Autism * Higher spare capacity in patient
Multiple acyl-CoA dehydrogenase deficiency (MADD) ** Higher spare capacity in patient
PLoS One. 2009 Dec 17; 4(12):e8329. Song Y et al
Cell type: Lymphocytes
Cell type: Fibroblasts
Cell type: Fibroblasts
Cell type: Fibroblasts
Patient Patient
Patient Patient
Data courtesy of James Bennet Lab University of Virginia
Data courtesy of Sher Hendrickson NCI/NIH Frederick
High Glycolytic Capacity and Impaired Oxidative Phosphorylation in Tumor cells
0%
20%
40%
60%
80%
100%
120%
140%
160%
0 0.008 0.04 0.2 1 5
Oligomycin (µM)
% o
f bas
elin
e ra
te
0%
20%
40%
60%
80%
100%
120%
140%
160%
% o
f con
trol A
TP le
vel
OCRECARATP%
of b
asel
ine
OCR
or
ECA
R
% o
f con
trol
ATP
leve
l
Oligomycin (µM)
H460 +glucose
A549
0%
20%
40%
60%
80%
100%
120%
140%
160%
0 0.008 0.04 0.2 1 5Oligomycin (µM)
% o
f bas
elin
e ra
te
0%
20%
40%
60%
80%
100%
120%
140%
160%
% o
f co
ntro
l ATP
leve
l
OCRECARATP%
of b
asel
ine
OCR
or
ECA
R
% o
f con
trol
ATP
leve
l
Oligomycin (µM)
A549 +glucose
0%
50%
100%
150%
200%
250%
0 0.8 4 20 50 100 2002-deoxyglucose (mM)
% o
f bas
elin
e ra
te
0%
50%
100%
150%
200%
250%
% o
f con
trol A
TP le
vel
OCRECARATP
0%
50%
100%
150%
200%
250%
0 0.8 4 20 50 100 2002-deoxyglucose (mM)
% o
f bas
elin
e ra
te
0%
50%
100%
150%
200%
250%
% o
f con
trol A
TP le
vel
OCRECARATP
% o
f bas
elin
e O
CR o
r EC
AR
% o
f con
trol
ATP
leve
l
2-Deoxyglucose (mM)
% o
f bas
elin
e O
CR o
r EC
AR
% o
f con
trol
ATP
leve
l
H460 A549
2-Deoxyglucose (mM)
Am J Physiol Cell Physiol; 2007 Jan
NEURODEGENERATION Spare Capacity Reveals More Than Basal Metabolism
0 24
Free Radic Biol Med. 2011 Apr 1; 50(7):866-873. Impaired spare respiratory capacity in cortical synaptosomes from Sod2 null mice Flynn J M, Choi S W, Day N U, Gerencser A A, Hubbard A and Melov S
BASELINE OLIGO FCCP ROT / MYX
Sod2 +/+
Cell type: Mouse CD-1 Synaptosomes
Endocrinology, February 2011 Dec:152(2):556-567 Medroxyprogesterone Acetate Antagonizes Estrogen Up-Regulation of Brain Mitochondrial Function Ronald W. Irwin, Jia Yao, Syeda S. Ahmed, Ryan T. Hamilton, Enrique Cadenas, and Roberta Diaz Brinton
Primary rat hippocampal neurons Primary rat mixed glia
NEURODEGENERATION Stress Test Reveals Link Between Cytosol and Hormone Therapy in Primary Neurons and Mixed Glia
Medroxyprogesterone [MPA] reduces the protective effect of estrogen [E2] in primary hippocampal neurons and mixed glia
E2 +MPA
E2 E2
E2 +MPA
Whole rat forebrain mitochondria
E2 E2+MPA
5 µM HNE 10 µM HNE
HNE
0 µM HNE Oligo
FCCP Antimycin A
0
50
100
150
250
300
0 25 50 75 100 125 150 175 200
OC
R (
% b
ase
lin
e)
Time (min)
200
Biochem J. 2009 Nov 15;425(1):99-107 Importance of the bioenergetic reserve capacity in response to cardiomyocyte stress induced by 4-hydroxynonenal Bradford G. Hill, Brian P. Dranka, Luyun Zou, John C. Chatham and Victor M. Darley-Usmar
CARDIOVASCULAR Stress Test Sensitivity Reveals Sub-Lethal Stress Response and Mechanism
XF Cell Mito Stress Test
20 µM HNE
Cell type: Neonatal Rat Ventricular Myocytes
STEM CELLS Stress Test Reveals Differentiation Status
J Cell Sci. 2011 Feb 1;124(Pt 3):348-58 A reduction in ATP demand and mitochondrial activity with neural differentiation of human embryonic stem cells Birket et al
Multipotent I6 neural stem cells Pluripotent I6 human embyronic stem cells
Differentiated I6 neural stem cells BJ fibroblasts
Human embryonic stem cells have higher levels of mitochondrial respiration and a greater proportion of ATP production through mitochondrial respiration
IMMUNOLOGY Spare Capacity Affects Memory T Cell Development and Survival
Immunity 2011 December 28 Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development van der Windt, JW, … and Pearce, EL, Washington School of Medicine
Naïve T cells Effector T cells Memory T cells
Cell type: Mouse Lymphocytes Spleen & Lymph Nodes
IMMUNOLOGY Spare Capacity in CD8+ T Cells is Dependent on Mitochondrial Fatty Acid Oxidation
Immunity 2011 December 28 Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development van der Windt, JW, … and Pearce, EL, Washington School of Medicine
Cell type: Mouse Lymphocytes Spleen & Lymph Nodes Fatty acid source is FBS
XF FAO Kit XF Palmitate-BSA FAO Reagent
Provides the first fully validated and standardized Palmitate-BSA FAO reagent:
• No substrate preparation • Activity tested • Quality controlled
SCREENING Drug Discovery and Toxicity Results from first pass screen of LOPAC library
(1,280) for mitochondrial biogenesis and toxicity
Cell type: Primary Rabbit Renal Proximal Tubule Cells (RPTC)
Anal. Biochem. (2010) Sept 1; 404(1):75-81 A high-throughput respirometric assay for mitochondrial biogenesis and toxicity C.C. Beeson et al.
Cisplatin HgCl2
SRT1720 AICAR Metformin
Trea
ted
FCC
P m
axim
al O
CR
/ Ve
hicl
e co
ntro
l
检测整体胰岛功能的胰岛捕获微孔板
Tissue micro-chamber (3 mm diameter, 200-350 µm high
Screen - 200 µm mesh
MODEL ORGANISMS Beyond Cells
Data courtesy of Auwerx Lab École Polytechnique Fédérale de Lausanne
Bioenergetic profiling of zebrafish embryonic development Stackley KD, Beeson CC, Rahn JJ, Chan SS. PLoS One. 2011 6(9):e25652
***
FCCP injection
basa
l
FCCP
basal FCCP
C.elegans Zebrafish
Global analysis of SUMO chain function reveals multiple roles in chromatin regulation Tharan Srikumar, Megan C. Lewicki, Michael Costanzo, Johnny M. Tkach, Harm van Bakel, Kyle Tsui, Erica S. Johnson, Grant W. Brown, Brenda J. Andrews, Charles Boone, Guri Giaever, Corey Nislow, Brian Raught. J. Cell Biol. Vol. 201 No. 1 145–163
Yeast