海馬生物能量測定儀 Seahorse XF analyzer
分析粒線體功能與 代謝研究的最佳工具
尚博生物科技公司
Office: 02-27855860 Mobile:0919830158 [email protected]
Product specialist 李文瑋David Li
Cell-Bio Biotechnology Co., Ltd.
尚博生物科技
Mitochondria
尚博生物科技
Decade of the mitochondria Decade of the mitochondria
Cancer Neurodegenerative
Disease
Cardiovascular Hepatobiology &
In vitrotoxicity Obesity/Diabetes
Aging
尚博生物科技
Legacies from Leland Clark
‧ End-point assay
‧ Single parameter
‧ Full user attention
‧ Low though-put
‧ High sample amount
1953 - Clark Electrode
O2 + 4 e− + 2 H2O →4 OH−
尚博生物科技
Cellular Bioenergetics for the 21st Century
Extracellular Flux (XF) Analyzers
尚博生物科技
Major Bioenergetics Pathway
Glycolysis
Kreb Cycle
CO
2ATP NADH
Pyruvate
NADH ATP
Electron Transport Chain
NAD+ NAD+
Lots of ATP
O
2Lactate
&
NADH NAD+
H+
Glucose
OCR,Oxygen
Consumption Rate
ECAR,
Extracellular Acidification Rate
尚博生物科技 Well 1 Well 2 Measuring Oxygen Consumption Rate (OCR) and Measuring Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR)
Extracellular Acidification Rate (ECAR)
Fiber-optic waveguide
Oxygen Fluorescent sensor Cells
Disposable probes
XF Culture Plate
尚博生物科技
Measuring Oxygen Consumption Rate (OCR) and Measuring Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR)
Extracellular Acidification Rate (ECAR)
Well 1 Well 2
The bio- cartridge is
raised, bringing the system back to baseline
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 (ECAR)
5 6 7
mm Hg (OCR)
The rate is calculated from the
slope
C2 C2 C2 C2
C2 C2 C2
C2
C2 C2C2
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
ECAR
OCR
A temporary micro- chamber is
formed
尚博生物科技
Quantifying Bioenergetics and Substrate Utilization More Aerobic
Less Energy
More Energy
More Glycolytic
ECAR (µpH/min)
OCR (pmole/min)
C6H12O6→2C3H4O3+ 2H++ 2ATP H+/ATP = 1, O2/ATP = 0
Glycolysis
+ +
+ +
+ +
+ + +
+ +
+ +
+ +
+
Glucose Oxidation
C6H12O6+ 6O2→6CO2+ 6H2O + 38ATP H+/ATP = 0.200, O2/ATP = 0.158
+ +
+ +
+
C16H32O2+ 23O2→16CO2+ 16H2O + 129ATP H+/ATP = 0.124, O2/ATP = 0.178
+ +
+
Palmitate Oxidation
+ +
Metabolic Inhibitors
+ +
+ +
+ + +
+ +
+ +
+ +
Uncoupler
+ +
+ +
+ + + +
+ +
+ +
+ +
+ +
+ + +
+ +
+ + +
+ +
+ + +
+ +
+ +
+ +
+
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4 or 2 Injection Ports/Well
Multiple Automatic Injection Ports Allows More Multiple Automatic Injection Ports Allows More Conditions to be Assessed Per Experiment
Conditions to be Assessed Per Experiment
Samples injected automatically
Samples injected automatically
0 100 200 300 400 500 600
0 50 100 150 200
Glycolysis (ECAR, mpH/min)
Aerobic Respiration (OCR, pmoles/min)
Less Energy
More Glycolytic More
Aerobic
More Energy
FCCP
Basal 2-DG
2-DG Rot + Rot
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Mitochondrial Function
尚博生物科技
Non-mitochondrial
Basal Resp.
ATP turnover
Respiratory Capacity
H+Leak
Increased Physiological Content in a Single Well
Oligomycin FCCP Rotenone
Nicholls Lab, Buck Inst., unpublished results 2008.
I II
III IV
V
Inner membrane
NADH
NAD+ FADH2
FAD
4H++O2
2H2O ADP+Pi
ATP
H+
H+ H+
H+
H+
尚博生物科技
In Vitro Assessment of Mitochondrial Dysfunction and Cytotoxicity as an indicator of potential toxicity
OCR
ECAR
Toxicol Sci 2008; 103 (2):335-45
尚博生物科技
Identifying compounds that cause oxidative stress Identifying compounds that cause oxidative stress
Primary human hepatocytes
+ 50 µM dantrolene
Primary human hepatocytes
+ 50 µM dantrolene + 10 µg/ml Oligomycin
ATP
ATP
ATP Drug Discov Today 2008; 13 (5-6):268-74
尚博生物科技
Demand Reserve Max Capacity
time
Demand Reserve Max Capacity
time
All cells carry an energy reserve that determines how they All cells carry an energy reserve that determines how they will handle an overload or chronic stress
will handle an overload or chronic stress
ATP DemandATP Demand Functional,
Healthy
Disease, Aging
J Neurochem 2009; 109:1179-91.
Biochem J 2009; in revision.
尚博生物科技
Oligo FCCP
3 independent +/+ mice preps
Oligo FCCP
3 independent -/- mice preps SOD
SOD-/-/-- shows dramatic decrease in respiratory capacityshows dramatic decrease in respiratory capacity
Nicholls Lab, Buck Inst, unpublished results 2009.
尚博生物科技
Modeling stochastic neuron degeneration:
The bioenergetic ‘hundred year flood’
J Neurochem 2009; 109:1179-91.
尚博生物科技
0 5 10 20 HNE (µM)
Actin Protein-HNE
Brad Hill et al., Biochem J 2009
MyocyteViability (%)
HNE (µM)
* * *
Protein
Protein--HNE modifications are associated with loss of HNE modifications are associated with loss of mitochondrial function and cell death
mitochondrial function and cell death
尚博生物科技 0 50 100 150 200 250 300
0 25 50 75 100 125 150 175 200
OCR (% baseline)
Time (min) 0 µM HNE
5 µM HNE 10 µM HNE 20 µM HNE
Spare respiratory capacity is an indicator of cell stress Spare respiratory capacity is an indicator of cell stress
Brad Hill et al., Biochem J 2009; accepted.
Rat neonatal ventricular myocytes
尚博生物科技
Mitochondria functional index
Basal respiration
– Drug toxicity and dose study Oxo-pho efficiency
– Identify toxicity mechanism Photon leak
– Structure integrity
– Aging, mutation, oxidative stress and drug Respiratory capacity- cellular stress
– Stress capacity
尚博生物科技
Substrate Utilization
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OCR % from baseline
A. Kinetics B. AUC
Time (Min)
Metformin Enhances FAO and Lowers Glucose Oxidation in C2C12 Myotubes
AUC OCR Induction
-500 500 1500 2500 3500 4500 5500 6500 untreated + BSA (veh cont.)
untreated + Glucose untreated + PA-BSA Metformin + Glucose Metformin + PA-BSA Metformin + BSA
Metformin (1mM) - - - + + +
Glucose (25mM) - + - + - -
Palmitate (150μM) - - + - + -
Wu Poster; GRC Molecular and Cellular Bioenergetics, 2007.
尚博生物科技
XF Assays can detect catabolism of exogenous and/or XF Assays can detect catabolism of exogenous and/or endogenous substrates
endogenous substrates
Johan AuwerxNature 2009; 458:1056-60
Palmitate Etomoxir
C2C12 myoblasts
C2C12 myoblasts
= Etomoxir
尚博生物科技
Islet Capture Microplate
尚博生物科技
“Increase in oxygen consumption with glucose in islets and clonal beta cells;
but not in C2C12 myoblasts”
Islets C2C12 myoblasts
Blank injection A; oligomycin at B 20mM glucose injection A;
oligomycin at B Oxygen consumption of Mice islets
Shirihai Lab, BUMC, unpublished results 2009.
尚博生物科技
Normal human islets Type 2 diabetic human islets
“Response to glucose blunted in diabetic islets ”
Blank injection A; oligomycin at B
20mM glucose injection A; oligomycin at B Oxygen consumption of Human islets
Shirihai Lab, BUMC, unpublished results 2009.
尚博生物科技
Substrate utilization
‧Metabolic Disease
‧Obesity
‧Diabetes
‧Metabolic regulator development
尚博生物科技
Energy Expenditure
尚博生物科技
The Connection Between Cancer and Glucose Metabolism
GLUT1
Glycolytic enzymes
Modified from: Kelloft GJ et. Al. Clinical Cancer Research 2005 11(8), 2785
“Progress and Promise of FDG-PET Imaging for Cancer Patient Management and Oncologic Drug Development.”
HIF- HIF -1 1α α
Prolyl
Prolyl hydroxylasehydroxylase
DMOG DMOG
“The prime cause of cancer is the replacement of the respiration of oxygen in normal body cells by a
fermentation of sugar.”
Otto Warburg 1883
1883 --19701970
Mitochondrial respiration
尚博生物科技
0%
20%
40%
60%
80%
100%
120%
140%
160%
0 0.008 0.04 0.2 1 5
Oligomycin (μM)
% of baseline rate
0%
20%
40%
60%
80%
100%
120%
140%
160%
% of control ATP level
OCR ECAR ATP
A549
Oligomycin(μM)
High Glycolytic Capacity and Impaired Oxidative Phosphorylation in Tumor cells
0%
50%
100%
150%
200%
250%
0 0.8 4 20 50 100 200 2-deoxyglucose (mM)
% of baseline rate
0%
50%
100%
150%
200%
250%
% of control ATP level
OCR ECAR ATP
A549
2-Deoxyglucose(mM)
Am J Physiol Cell Physiol 2007; 292:C125-36.
尚博生物科技
Hereditary Leiomyomatosis and Renal Cell Cancer Model:
Glycolytic Flux in FH/LDH-A deficient cells Mol Cancer Ther 2009; 8(3):626-635.
尚博生物科技
LDH-A inhibition results in increased apoptosis via ROS production in FH knockdown cell line
Mol Cancer Ther 2009; 8(3):626-635.
尚博生物科技
Basal rates of various cell types determined by type of cell, media and environmental conditions
HepG2 CHO-M3 H460
C2C12 Tubes
C2C12 Blasts HELA-BH3I
SK-Hep1 SHSY5Y A549
尚博生物科技 細胞健康管理
幫細胞把脈
對品質把關
尚博生物科技
Cell quality check of primary cardiomyocytes identifies functional cells
Red = “healthy”
Blue = “not healthy”
Data courtesy of Craig Beeson, MUSC
尚博生物科技
ES cell quality check and differentiation study
Highly glycolytic
Specific metabolic
profile
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Day 7 C2C12 Myocytes Grown Under Different Conditions
• Insulin sensitive
• Good glucose utilization
• Very responsive to exogenous fat
• Insulin insensitive
• Poor glucose utilization
• Low response to exogenous fat
More Aerobic
More Energetic
Less
Energetic More Anaerobic
More
Aerobic More Energetic
Less
Energetic More Anaerobic
Obese Model Lean Model
Determining optimal cell culture conditions to mimic appropriate biology
尚博生物科技 Summary
非接觸式光學探針設計 自動化操作與藥物注射系統 快速檢測
– 粒線體功能 – 代謝系統運作
四不一沒有
– 不用額外試劑 – 不需破壞樣本 – 不用接觸細胞 – 不用人力監控 – 沒有學習門檻 Seahorse XF 96 analyzer
尚博生物科技 sunmmary
‧Mitochondrial functional index
– Basal respiration – Oxo-pho efficiency
– Photon leak- structure integrity – Respiratory capacity- cellular stress
‧Substrate utilization
– Sugar, fat and amino acid
‧Energy expenditure
– Aerobic or anaerobic pathway – 細胞把脈技術
尚博生物科技
尚博生物科技
Cell-bio biotechnology Office: 02-27855860 Mobile:0919830158 [email protected]
Product specialist 李文瑋David Li
