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Difference between revisions of "Fasching IOC61-TPP group"

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{{Abstract
{{Abstract
|title=Fasching M, Sumbalova Z (2011) TPP special interest group during IOC61
|title=Fasching M, Sumbalova Z (2011) TPP special interest group during IOC61. MiPNet16.01.
|info=[http://www.oroboros.at/index.php?id=ioc61 IOC61]
|info=[[IOC61|IOC61]]
|authors=Fasching M
|authors=Fasching M, Sumbalova Z
|year=2011
|year=2011
|event=IOC61
|event=IOC61
|abstract=The simultaneous measurement of'''[[Mitochondrial membrane potential]]''' with the TPP method and respiration under HRR conditions with the OROBOROS ISE system is explained, practiced and discussed in a "hands-on" approach.
|abstract=The simultaneous measurement of '''[[Mitochondrial membrane potential]]''' with the TPP method and respiration under HRR conditions with the Oroboros ISE system is explained, practiced and discussed in a "hands-on" approach.
|mipnetlab=AT Innsbruck OROBOROS
|mipnetlab=AT Innsbruck Oroboros , SK Bratislava Sumbalova Z
}}
}}
{{Labeling}}
{{Labeling}}
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'''TPP special interest group during IOC61'''  
'''TPP special interest group during IOC61'''  


'''[[High-resolution respirometry]]''' (HRR) provides a quantitative approach to bioenergetics and mitochondrial physiology with the [[Oxygraph-2k]] (O2k; OROBOROS INSTRUMENTS) offering several sole-source features.
During IOC61 Oroboros Instruments offers a [[TPP special interest group during an International Oxygraph Course]] covering the utilization of the [[O2k-TPP+ ISE-Module|Oroboros ISE system]] to measure [[mitochondrial membrane potential]]. Some topics of general interest will be covered together with the participants from the "main course".  
 
This special interest groups is intended as a workshop, therefore the exact program will depend on the level of experience and interests of the participants. In an introductory session the principals of measuring mitochondrial membrane potential with a TPP-electrode are covered. The different steps of planning and performing a TPP are discussed. In "hands-on" sessions the participants will follow [[MiPNet15.03_O2k-MultiSensor-ISE]] to assembly TPP and reference electrodes, set up an oxygraph-2k with these electrodes, record an instrumental oxygen background in their presence, calibrate the electrodes and determine the effect chemicals used in a biological experiment on the electrodes.
Hardware and software developments are based on long-term expertise with [[polarographic oxygen sensor]]s (POS) [1] and continuous evaluation relative to alternative sensors. The POS is superior in the range from zero oxygen to pure oxygen at about 1 mM dissolved O<sub>2</sub>, yielding a 500,000-fold dynamic range with a digital resolution of 2 nM in the O2k [2]. [[Oxygen flux]] is measured in the [[closed system]] as the negative time derivative of [[oxygen concentration]], calculated and displayed on-line with correction for [[instrumental background oxygen flux]], yielding a resolution of 1 pmol O<sub>2</sub>.s<sup>-1</sup>.ml<sup>-1</sup>. Minimization, experimental evaluation, and automatic correction (DatLab) of instrumental background oxygen flux are integral to HRR [2], as emphasized in practical tests and data analysis during the basic [[O2k-Workshop]]. Alternatively, oxygen flux can be measured in an [[open system]] mode of operation, using the [[Titration-Injection microPump]] (TIP2k) for feedback control of oxygen levels by matching oxygen supply to demand, particularly at graded levels of [[hypoxia]] in studies of [[oxygen kinetics]] [3].
In a hands-on experiment this set up will be used in the experiment with tissue homogenate from mouse brain for measurement of changes in mitochondrial membrane potential simultaneously with changes in respiration in the course of [[Substrate-uncoupler-inhibitor titration|SUIT]] protocol. [[Calculation of mitochondrial membrane potential from measurements with a TPP electrode]] will be intensively discussed and facilitated by the provided [[Spreadsheet templates| spreadsheet templates]].  
 
Specificity of several SUIT protocols will be discussed.
Most applications of HRR take advantage of the high stability and sensitivity of the O2k in [[phosphorylation control protocol]]s with intact cells, or [[substrate-uncoupler-inhibitor titration]] (SUIT) protocols with [[isolated mitochondria]], [[permeabilized cells]] or tissues, in particular permeabilized muscle fibres [4]. Physiological temperatures (electronic Peltier control, within 0.001 °C), optimized incubation media ([[MiR06]]), and a rationale for the design of tested SUIT protocols [5] are the hallmark of quantitative and comparative mitochondrial respiratory physiology. [[Coupling control]] and [[substrate control]] of mitochondrial respiration are expressed as [[flux control ratio]]s, resolving some confusion related to the [[respiratory control ratio]] (RCR) [6,7]. Applicaton of [[CI+II]] substrate combinations in SUIT protocols extends conventional bioenergetic studies to the level of mitochondrial physiology, some principles of which are applied and discussed at the O2k-Workshop, whereas an in-depth introduction is provided at the [[MiPsummer 2011|MiP''summer'' School]].
Interested participants can join a special group combining HRR with measuring [[Mitochondrial_membrane_potential#Mitochondrial_membrane_potential_of_permeabilized_fibres|Mitochondrial membrane potential of permeabilized fibres]].
 
Compared to the long tradition of applications of polarographic oxygen sensors, replacing the classical manometric (Warburg) apparatus, HRR is a recent development, which now provides a widely applied tool for routine and specific analyses of mitochondrial function/dysfunction where ''(1)'' reliability and quality control are important (clinical studies, functional diagnosis), ''(2)'' the amount of biological material is limited (<0.5 mill cultured cells, 1-2 mg of fresh tissue from biopsies; <0.05 mg of mitochondrial protein), ''(3)'' pathological effects result in reduced respiration, and ''(4)'' effects need to be tested at physiological, low intracellular oxygen levels [8].
 
Finally, the basic O2k-Workshop provides an overview on [[O2k-MultiSensor System]] applications, for the simultaneous measurement of respiration and [[mitochondrial membrane potential]] (TPP<sup>+</sup> electrode), acidification (pH electrode), nitric oxide (amperometric), spectrophotometry (cytochrome spectra), and spectrofluorimetry (Amplex red, safranin, etc.). In parallel sessions, a hands-on introduction is provided to the application of the TPP<sup>+</sup> electrode for advanced users [9]. Taken together, HRR integrates metabolic and physicochemial concepts on fluxes and forces in open and closed systems [10].
 
# [[Gnaiger_1983_POS|Gnaiger E, Forstner H, eds (1983) Polarographic Oxygen Sensors. Aquatic and Physiological Applications. Springer, Berlin, Heidelberg, New York: 370 pp.]]
# [[Gnaiger_2008_POS|Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley: 327-352.]]
# [[Gnaiger_2001_RespPhysiol|Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir. Physiol. 128: 277-297.]]
# [[Pesta_2010_Protocols|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.]]
# [[Gnaiger_2007_MitoPathways|Gnaiger E ed (2007) Mitochondrial Pathways and Respiratory Control. OROBOROS MiPNet Publications, Innsbruck: 96 pp.]] - Electronic 1st ed: http://www.oroboros.at/index.php?mipnet-publications
# [[Gnaiger_2009_IJBCB|Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int. J. Biochem. Cell Biol. 41: 1837-1845.]]
# [[MiPNet12.15_RespiratoryStates|Gnaiger E. MitoPathways: Respiratory states and flux control ratios. Mitochondr. Physiol. Network 12.15.]]
# [[Gnaiger_2000_PNAS|Gnaiger E, Méndez G, Hand SC (2000) High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. Proc. Natl. Acad. Sci. USA 97: 11080-11085.]]
# [[MiPNet14.05_TPP-MitoMembranePotential|Renner-Sattler K, Fasching M, Gnaiger E. TPP+ and membrane potential. Mitochondr. Physiol. Network 14.05.]]
# [[Gnaiger_1993_PAC|Gnaiger E (1993) Nonequilibrium thermodynamics of energy transformations. Pure Appl. Chem. 65: 1983-2002.]]
 
[[Category:IOC61]]

Latest revision as of 11:02, 23 January 2019

Fasching M, Sumbalova Z (2011) TPP special interest group during IOC61. MiPNet16.01.

Link: IOC61

Fasching M, Sumbalova Z (2011)

Event: IOC61

The simultaneous measurement of Mitochondrial membrane potential with the TPP method and respiration under HRR conditions with the Oroboros ISE system is explained, practiced and discussed in a "hands-on" approach.


• O2k-Network Lab: AT Innsbruck Oroboros, SK Bratislava Sumbalova Z


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TPP special interest group during IOC61

During IOC61 Oroboros Instruments offers a TPP special interest group during an International Oxygraph Course covering the utilization of the Oroboros ISE system to measure mitochondrial membrane potential. Some topics of general interest will be covered together with the participants from the "main course". This special interest groups is intended as a workshop, therefore the exact program will depend on the level of experience and interests of the participants. In an introductory session the principals of measuring mitochondrial membrane potential with a TPP-electrode are covered. The different steps of planning and performing a TPP are discussed. In "hands-on" sessions the participants will follow MiPNet15.03_O2k-MultiSensor-ISE to assembly TPP and reference electrodes, set up an oxygraph-2k with these electrodes, record an instrumental oxygen background in their presence, calibrate the electrodes and determine the effect chemicals used in a biological experiment on the electrodes. In a hands-on experiment this set up will be used in the experiment with tissue homogenate from mouse brain for measurement of changes in mitochondrial membrane potential simultaneously with changes in respiration in the course of SUIT protocol. Calculation of mitochondrial membrane potential from measurements with a TPP electrode will be intensively discussed and facilitated by the provided spreadsheet templates. Specificity of several SUIT protocols will be discussed. Interested participants can join a special group combining HRR with measuring Mitochondrial membrane potential of permeabilized fibres.

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