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Difference between revisions of "Pesta 2012 Methods Mol Biol"

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Standardized two-point calibration of the polarographic oxygen sensor (static sensor calibration), calibration of the sensor response time (dynamic sensor calibration), and evaluation of instrumental background oxygen flux (systemic flux compensation) provide the unique experimental basis for high accuracy of quantitative results and quality control in highresolution respirometry.
Standardized two-point calibration of the polarographic oxygen sensor (static sensor calibration), calibration of the sensor response time (dynamic sensor calibration), and evaluation of instrumental background oxygen flux (systemic flux compensation) provide the unique experimental basis for high accuracy of quantitative results and quality control in highresolution respirometry.
|keywords=Substrate-uncoupler-inhibitor titration, Human vastus lateralis, Needle biopsy, HEK, HPMC, HUVEC, Fibroblasts, Routine respiration, Oxidative phosphorylation, Q-junction, Pyruvate, Glutamate, Malate, Succinate, Leak, Coupling control, Uncoupling, Oxygraph, Oxygen flux, Residua oxygen consumption, Instrumental background
|keywords=Substrate-uncoupler-inhibitor titration, Human vastus lateralis, Needle biopsy, HEK, HPMC, HUVEC, Fibroblasts, Routine respiration, Oxidative phosphorylation, Q-junction, Pyruvate, Glutamate, Malate, Succinate, Leak, Coupling control, Uncoupling, Oxygraph, Oxygen flux, Residua oxygen consumption, Instrumental background
|info=[http://www.oroboros.at/fileadmin/user_upload/Reprints/Pesta_2010_Protocols.pdf pdf]
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Revision as of 10:19, 16 September 2010

Publications in the MiPMap
Pesta D, Gnaiger E (2010) High-Resolution Respirometry. OXPHOS protocols for human cell cultures and permeabilized fibres from small biopisies of human muscle. In: Mitochondrial bioenergetics: methods and protocols (Series Editor: Sir John Walker), edited by Carlos Palmeira and Antonio Moreno. In press.

Β» pdf

Pesta D, Gnaiger E (2010)

Abstract: Protocols for high-resolution respirometry of intact cells, permeabilized cells, and permeabilized muscle fibres offer sensitive diagnostic tests of integrated mitochondrial function using standard cell culture techniques and small needle biopsies of muscle. Multiple substrate-uncoupler-inhibitor titration (SUIT) protocols for analysis of oxidative phosphorylation improve our understanding of mitochondrial respiratory control and the pathophysiology of mitochondrial diseases. Respiratory states are defined in functional terms to account for the network of metabolic interactions in complex SUIT protols with stepwise modulation of coupling and substrate control. A regulated degree of intrinsic uncoupling is a hallmark of oxidative phosphorylation, whereas pathological and toxicological dyscoupling is evaluated as a mitochondrial defect. The non-coupled state of maximum respiration is experimentally induced by titration of established uncouplers (FCCP, DNP), to collapse the proton gradient across the mitochondrial inner membrane and measure the capacity of the electron transfer system (ETS, open-circuit operation of respiration). Intrinsic uncoupling and dyscoupling are evaluated as the flux control ratio between non-phosphorylating LEAK respiration (electron flow coupled to proton pumping to compensate for proton leaks) and ETS capacity. If OXPHOS capacity (maximally ADP stimulated oxygen flux) is less than ETS capacity, the phosphorylation system contributes to flux control. Physiological Complex I+II substrate combinations support maximum ETS and OXPHOS capacities, due to the additive effect of multiple electron supply pathways converging at the Q-junction. Substrate control with electron entry separately through Complex I (pyruvate+malate or glutamate+malate) or Complex II (succinate+rotenone) restricts ETS capacity and artificially enhances flux control upstream of the Q-cycle, providing diagnostic information on specific branches of the ETS. Oxygen levels are maintained above air saturation in protocols with permeabilized muscle fibres to avoid experimental oxygen limitation of respiration. Standardized two-point calibration of the polarographic oxygen sensor (static sensor calibration), calibration of the sensor response time (dynamic sensor calibration), and evaluation of instrumental background oxygen flux (systemic flux compensation) provide the unique experimental basis for high accuracy of quantitative results and quality control in highresolution respirometry. β€’ Keywords: Substrate-uncoupler-inhibitor titration, Human vastus lateralis, Needle biopsy, HEK, HPMC, HUVEC, Fibroblasts, Routine respiration, Oxidative phosphorylation, Q-junction, Pyruvate, Glutamate, Malate, Succinate, Leak, Coupling control, Uncoupling, Oxygraph, Oxygen flux, Residua oxygen consumption, Instrumental background

β€’ O2k-Network Lab: AT_Innsbruck_GnaigerE


Labels:

Stress:Hypoxia  Organism: Human  Tissue;cell: Skeletal Muscle"Skeletal Muscle" is not in the list (Heart, Skeletal muscle, Nervous system, Liver, Kidney, Lung;gill, Islet cell;pancreas;thymus, Endothelial;epithelial;mesothelial cell, Blood cells, Fat, ...) of allowed values for the "Tissue and cell" property.  Preparation: Intact Cell; Cultured; Primary"Intact Cell; Cultured; Primary" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property., Permeabilized Cell or Tissue; Homogenate"Permeabilized Cell or Tissue; Homogenate" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property. 

Regulation: Respiration; OXPHOS; ETS Capacity"Respiration; OXPHOS; ETS Capacity" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property., Flux Control; Additivity; Threshold; Excess Capacity"Flux Control; Additivity; Threshold; Excess Capacity" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property., Coupling; Membrane Potential"Coupling; Membrane Potential" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property., Mitochondrial Biogenesis; Mitochondrial Density"Mitochondrial Biogenesis; Mitochondrial Density" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property., Substrate; Glucose; TCA Cycle"Substrate; Glucose; TCA Cycle" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property. 


HRR: Oxygraph-2k, TIP2k, Chemicals; Media"Chemicals; Media" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property., Method"Method" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property.