Mitochondria

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Mitochondria
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Measuring oxygen consumption of pancreatic islets
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Glucose induced Insulin secretion and Oxygen
consumption correlate
Longo et al 1991, JBC
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Pre-transplantation islet quality assessment
1000-2000 islets
Papas et al 2007, American J Transplantation
Sweet et al 2008, American J Transplantation
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Measuring oxygen consumption in islets
Challenges -Primary tissue -Sample
variability and vulnerability -Non
adherent Current methods -Perifusion systems
Coated microplates -Low throughput -User
dependent -Time consuming -Labor intense -Large
data variability
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Islet plate development
Well in a 24-well plate
Standard Seahorse plate
Islet plate
Top view
Depression keeps islets in center Screen
decreases turbulence exposure
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Top view islets behind bars
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Seahorse principle
Small volume makes O2 drop
Algorithm translates drop in O2 to oxygen
consumption rates
Oligomycin
Oligomycin
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H
Oligomycin Decrease respiration
Rotenone Decrease respiration
FCCP Increase respiration
Myxothiazol Decrease respiration
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Oxygen consumption of Mice islets
Stable oxygen consumption over several hours
Blank injections at A B
Blank injection A oligomycin at B
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Oxygen consumption of Mice islets
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
INS1 beta cells
20mM glucose injection A oligomycin at B
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Oxygen consumption of Human islets
Response to glucose blunted in diabetic islets
Normal human islets
Type 2 diabetic human islets
Blank injection A oligomycin at B
20mM glucose injection A oligomycin at B
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Oxygen consumption of Mice islets
High level of uncoupled respiration in islets
Absolute units
Normalized to initial measurements
Islets
Islets
C2C12 myoblasts
C2C12 myoblasts
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Islet plate protocol overview
Day1 Isolation of islets, culture overnight
or multiple days depending on experiment
Day2 Preparation of injection compounds, loading
into injection ports Program
experiment in XF24 software
Day2 Transfer islets to Seahorse experiment
media with 1 serum Count islets in
microscope by taking aliquots of suspended islets
Seed islets by concentration, 70
islets/well Transfer screens to wells
Incubate islet plate at least 60min at
37ºC before experiment starts
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Boston University School of Medicine,
Boston Gilad Twig Shana Katzman Solomon
Graf
Boston University Barbara Corkey Buck
institute David Nicholls NASA Dan Barry
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