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Difference between revisions of "MiPNet15.08 TPP electrode"

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== High-resolution respirometry and mt-membrane potential ==
The O2k-MultiSensor system provides a potentiometric and a fluorometric module for measurement of the mt-membrane potential.
See [http://www.oroboros.at/?O2k-multisensor-manual O2k-MultiSensor Manual] for up-to-date information and discussions on the measurement of mtMP. The general manual for the OROBOROS Ion Selective Electrode system ([[ISE]] System) is in [[MiPNet15.03]]. While working with the potentiometric (pX) channel of the O2k, the general [[ESD|guidelines for avoiding damage to the oxygraph by ESD]] should be followed.
A rudimentary protocol for measuring mitochondrial membrane potential (MiPNet14.05), its mathematical appendix and the most up-to-date spreadsheet templates, DatLab templates, and DatLab demo files, can be found [http://www.oroboros.at/?Protocols_tpp-membranepotential here].
Before measuring membrane potential via [[TPP+]] is even started, the [[TPP+ inhibitory effect]] in the studied system should be explored.How to get from the measured TPP<sup>+</sup> concentration to the mitochondrial membrane potential: [[Calculation of mitochondrial membrane potential from measurements with a TPP electrode]]. This covers also the influence of [[Unspecific binding]].
Practical difficulties in applying the method to permeabilized fibers are discussed in [[Mitochondrial_membrane_potential#Mitochondrial_membrane_potential_of_permeabilized_fibres|Mitochondrial membrane potential of permeabilized fibers]]. Some tips for [[Cleaning the TPP+ electrodes]]Some important performance parameters of the TPP electrode are summarized on this [http://www.oroboros.at/fileadmin/user_upload/MiP2010/MiP2010_Sumbalova_poster1.pdf poster]. In any way we hope you will join us for one of our [[TPP special interest group during an International Oxygraph Course]].
=== Mitochondrial membrane potential and anoxia ===
=== Question ===
Can I use anoxia as a reference state for zero (minimum) mt-membrane potential in isolated mitochondria?Β  I want to run experiments in series without loosing the time for washing out inhibitors or uncouplers.Β  The protocol includes substrates and ADP.
=== Answer ===
Anoxia should provide a good reference value for minimum mt-membrane potential. However, you should carry out a test experiment: After reaching anoxia, add oligomycin as a test for the possibility that ATPsynthase acts as a ATPase and thus maintains a mt-membrane potential in reversed mode of operation.Β  Then titrate uncoupler (FCCP) to collaps the mt-membrane potential under anoxia.
Careful: Ethanol as a carrier for oligomycin and FCCP exerts a chemical side effect on the TPP+ signal, which has to be evaluated in a separate control experiment and subtracted from the experimental trace.
== Mitochondrial membrane potential of permeabilized fibres ==
=== General ===
From experiments with isolated mitochondria or permeabilized cells one can derive the concentration and mitochondrial activity (oxygen flux per volume) necessary to obtain reliable signals with the TPP<sup>+</sup> electrode. Since unspecific binding is higher om [[Permeabilized muscle fibre|permeabilized muscle fibers]] compared to isolated mitochondria, the amount of fibres should be chosen to obtained rather high volume-specific oxygen fluxes. Even with permeabilized cells higher sample concentrations are required, as compared with standard high-resolution respiratory measurements. It is important that the total amount of TPP in the chamber is known at all times. Therefore, the sample should not be preconditioned outside of the chamber to TPP, and even a rough estimation of the sample volume will be necessary.
=== Introduction of the sample ===
The established way to measure mitochondrial membrane potential for isolated mitochondria (and permeabilized cells) is to calibrate the TPP electrode by adding TPP in several steps to the experimental chamber. With the final calibration step the starting TPP<big>+</big> concentration is reached. Then the sample is injected into the "calibrated" chamber. Therefore, unlike in the application of other potentiometric methods (pH, Ca<sup>2+</sup>,..) the "calibration" does in fact serve two different purposes:
:1. Calibration of the sensor;
:2. Establishing the total amount of TPP in the chamber. This amount has to be precisely known for calculation of mtMP from the measured [TPP<sup>+</sup>].
==== Problems ====
Introduction of the sample is a key problem in extending the TPP<sup>+</sup> method for measuring mitochondrial membrane potential for two different reasons:
: (a) Disturbance of the calibration itself: That is after the necessary handling steps the calibration parameters determined during the calibration run are no longer correct (due to geometry changes, ...)
: (b) Changing the total amount of TPP preset in the chamber: If solution is lost or additional liquid has to be added during the introduction of the sample the information about the total amount of TPP+ may easily get lost:
Even for permeabilized cells, this was a major issue and was solved by injection a quite concentrated sample solution as fast as possible into the chamber: A fast injection ensured that the replacement of TPP containing medium in the chamber by the medium containing sample but no TPP+ can be treated as a simple process, not involving:
: 1. Any mixing of the solutions before the displacement is complete.
: 2. Any uptake of TPP by the sample before the displacement is complete. Therefore the introduction could be treated as a simple dilution of a solution with a known TPP conc. with a solution containing no TPP. (see also Note 1)
All methods to introduce a sample have to consider both problems.
==== Possible methods ====
: 1. The obvious method: '''opening the chambers''' and placing the permeabilized fiber in the chamber:
From very few and insufficient trials at Oroboros it would seem that problem I.) (loss of calibration information) is maybe less severe than initially thought or is at least less significant that problem B.). This could be checked by practicing the opening and closing the chamber with the introduction of any biological material. Typical opening and closing the camber will result in loss of liquid, necessitate the introduction of new liquid. To circumvent Problem B (change of TPP+ conc.) even in such a blind trial it would be necessary to replace all liquid lost with medium containing exactly the TPP concentration established din the chamber before opening it. However Problem B might proof crucial: After introducing permeabilized fibers they immediately start to take up TPP. Opening and closing the chamber typically requires quite a lot of β€œbubble fighting” and placing liquid on top of the stopper. While (pre-warmed) medium containing exactly the initial TPP+ conc. after calibration can be used for this operations, the concentration in the chamber will already be different bat this time due to TPP uptake. The problem will be the more significant the longer this operations continue and the more liquid is moved around.
: 2. Introduction of the sample via '''a dedicated large additional port''': Such a port would have to be closed during operation and calibration by a plug completely filling the bore (a plug e.g. only filling the top part of the stopper would create a huge unstirred zone inside the stopper, incompatible with high resolution respirometry). The construction of such a stopper that ensures a tight fit might pose some technical problems but is probable doable. Operation could work in the following way:
* End of calibration run, top of stopper is dry
* Plug for β€œsample port” is removed: since top of stopper is dry no additional liquid gets into the chamber
* Sample is introduced using ?very special forceps?,Β  ?a biopsy needle ?,Β  ?a steel wire? (volume of sample introduced should be known or very small)
* To enable bubble free closing: A very small volume (just as much as necessary) volume of pre warmed medium is immediately filed into the chamber via the β€œsample” port and the sample port is immediately closed with the plug. A small volume will be extruded from the chamber via the injection port. Because the sample already started to take up TPP+, the concentration of the volume extruded will not be exactly known (the volume will ideally be the sample volume + the volume added before closing the port).Β  The volume should be kept as small as possible to minimize the error.
This method requires a special stopper and plug, the handling procedure for introducing the sample has to be very special because the diameterΒ  of the β€œsample port” necessarily has to be quite small (there is just not too much space left) and introduced new problems regarding the design and closing procedure of the fitting plug. The advantage is that it is not necessary to move the electrodes.
: 3. Using the '''existing ports''' for an approach similar to the one discussed under point two. Depending on the method found to insert the sample into the chamber, either the '''reference electrode''' or the '''TPP electrode''' would be removed from the stopper (try top). The further process would be as described above.Β  The closing of the bore (with the electrode) is already a quite established process. If it is possible to use the port of the reference electrode, a very small (or considering the sample volume: no) additional solution (pre-warmed containing the initial TPP concentration as a first order approximation) can be used.
Advantage: No modification of stopper required, same development of β€œintroduction skills” necessary as for method 2. Same advantages / disadvantages in regard to β€œProblem B” as for Method 2
Disadvantage: possible disturbance of the calibration by moving one electrode. At least for removing and re-inserting the reference electrode this problem seems (from limited experience) to be quite small. This can be easily tested. Removal and reinsertion of electrodes should be done with stirrers switched off.
: 4. Using a '''partially homogenized sample''', injecting it: probable not doable with current (and possible) permeabilization protocols.
: 5. '''Preferred method:''' Introduction of the sample via the '''titration port''': Requires obviously the most sophisticated tool for getting the sample in, but only minimal distortion.
==== Evaluation and solution by the Neufer Group at East Carolina University ====
The group of [[US NC Greenville Neufer PD|Darrell Neufer at East Carolina University, Greenville, NC, USA]], evaluated two of the discussed approaches and presented a solution. For their full contribution see the Discussion page. In summary
* the port for the reference electrode is used to introduce the sample
* the sample is split into several parts
* if necessary, the sample pieces are introduced into the chamber using a standard Hamilton syringe and the reference electrode itself
* other methods for sample insertion were tested and rejected
'''Until full integration of their method into this page see the contribution of the Neufer group on the [[Talk:Mitochondrial_membrane_potential|Discussion page]] for a detailed description.'''
==== Method used by the OROBOROS O2k-Team ====
The method described above was further refined by the OROBOROS O2k-Team in Innsbruck using a glass Pasteur pipette to introduce the sample into the chamber via the port for the reference electrode. For full details see the [[Talk:Mitochondrial_membrane_potential|Discussion page]].
=== Performing the measurement ===
==== Reoxygenation and high oxygen ====
The method recommended by Oroboros Instruments to do a re-oxygenation in the presence of additional electrodes is to inject H2O2 into a medium containing catalase, avoiding any mechanical disturbances, see the protocol for the MiR06 medium [http://www.oroboros.at/index.php?id=protocols_miro6 MiPNet14.13]. However, if the presence of catalase in the medium is not desired or the necessary increase in oxygen concentration is larger than recommended for the MiR06 approach ( Ξ”cO2 ≀200 ΞΌmol/l) the method decribed by the Neufer group ion the discussion section seems to be an alternative.
Because the H2O2 method is limited to a delta cO2 of 200 Β΅mol/l the initial high oxygen concentration should be achieved with the gas opahse method before the statr of the experiment. The O2 level can then be maintained by H2O2 injections without opening the chamber.
==== Slowness of TPP uptake /release ====
TPP uptake and release seems generally to be slower for permeabilzed fibers than for isolated mitochondria or permeabilized cells. However, the extend of this effect was reported to be very different by different groups. It is not yet clear what causes extremely slow uptake/ release in some cases but not in others.
== Calculation of mitochondrial membrane potential from measurement of TPP<sup>+</sup> ==
Based on information provided in the O2k-Protocols [[MiPNet14.05 TPP-MitoMembranePotential |MiPNet 14.05]], which should be consulted first. The most up to date spreadsheet templates, DatLab templates, DatLab demo files, MiPNet14.05 and its mathematical appendix can be found [http://www.oroboros.at/index.php?protocols_tpp-membranepotential here].
The calculation ofΒ  mitochondrial membrane potential from measurements with a TPP electrode is a difficult and far from settled topic.
=== Sensitivity analysis of the method ===
[[File:Error_evaluation_absolute_1pc.png|thumb|300px|alt=absolute error in delta Psi by introduction of a 1 % error in c(TPP) plotted against delta Psi|absolute error in delta Psi by introduction of a 1 % error in c(TPP) plotted against delta Psi]]
The sensitivity of the method to small errors is strongly dependent on the membrane potential. For low membrane potential the method is inherently unsuitable. This is illustrated by introducing an artificial errors in the measured [[TPP]]+ concentration and plotting the resulting errors in the calculated membrane potential against the (original) membrane potential. The exact shape of the function depends on sample amount and type, binding correction and all other external factors but the general shape is usually quite constant.
Here this is illustrated for isolated (un-purified) mitochondria, simulating the effect of a +1% and -1% error in the measured TPP+ concentration.
The used calculation template is not able to deal properly with results that would lead to a negative membrane potential, therefore errors leading to a 0 or negative membrane potential are shown here as "zero".
=== Unspecific binding ===
==== The four compartment model ====
The approach to unspecific binding chosen in MiPNet 14.05 and in the OROBOROS Spreadsheet temples is basically based on Rottenberg's <ref name ="Rottenberg1984">Rottenberg H (1984) Membrane potential and surface potential in mitochondria: uptake and binding of lipophilic cations. J Membr Biol 81:127-38.</ref> 4 compartment model, developed for isolated mitochondria. As shown in the mathematical appendix to [[MiPNet 14.05]] this approach seems to be mathematically fundamentally equivalent to the approaches by Brand <ref name="Brand1995">Brand MD (1995) Measurement of mitochondrial protonmotive force. In: Bioenergetics a practical approach (Brown GC, Cooper CE, eds):39-62. Oxford University Press, Oxford.</ref> and Kamo <ref name="Demura1987">Demura M, Kamo N, Kobatake Y (1987) Binding of lipophilic cations to the liposomal membrane: thermodynamic analysis. Biochim Biophys Acta 903:303-8.</ref>, at least for the processes inside the mitochondria.
In a nutshell, 4 different compartments are considered:
: A: The liquid filled matrix of the mitochondria, containing β€œfree, internal” TPP+.
: B: Material (membranes etc ) exposed to the typically high TPP+ concentration in compartment A. In Rottenbergs original approach this is the inside face of the inner mitochondrial membrane.
: C: The liquid filled space outside the mitochondria. This comprises the entire volume of the sample chamber with the exception of compartments A, B, and D.
: D: Material (membranes etc) that are exposed to the typically low TPP+ concentrations outside the mitochondrial matrix. In Rottenberg's original approach this compartment comprises the outside face of the inner mitochondrial membrane and any present material from the outer mitochondrial membrane or traces of cell material not removed during purification.
The probe ion is supposed to accumulate in compartments B and D directly proportional to
* the β€œsize/amount” of the compartment, measured by some marker,Β  e.g. protein content
* the concentration of probe molecule in the adjunct liquid phase, e.g the TPP+ concentration in the mitochondrial matrix
* a factor describing the affinity of the compartment to the probe molecule (the binding correction factor.
E.g. the amount of TPP+ "bound" by the inward facing side of the inner mitochondrial membrane is
''n''(int,bound) = ''K''i' * Pmt * ''C''(int,free)
(Equation A8a in the mathematical Appendix to MiPNet14.05)
It should be noted that any binding correction factor (e.g. ''K''i’)is only useful together with a certain type of marker (Pmt) for which it was determined.
The approaches by Brand and Kamo do not consider the outside compartments for unspecific binding. Indeed, for purified isolated mitochondria the outside binding seems to have a very small effect. Therefore in all further considerations one has to discern between studies of purified isolated mitochondria and studies with other sample types.
=== Isolated mitochondria and unspecific binding in the mitochondria ===
Due to the small amount of material exposed to the outside concentrations and the low outside concentrations only the inside binding is significant.
The absolute values for delta Psi will depend on the chosen binding correction factors.
The absolute DIFFERENCE between membrane potentials (either between different states or different samples) will NOT depend on the chosen inside binding correction factor, see MiPNet 14.05 Mathematical Appendix. Therefore, it should be possible to obtain absolute change of delta Psi’s for this sample type. The insensitivity against outside binging can be shown by varying the outside binding parameter only:
[[File:Isolated mito Kout variation.png|500px|alt=various delta PSi values and one delta delta Psi values plotted against varying external binding parameter Kout'|various delta PSi values and one delta delta Psi values plotted against varying external binding parameter Kout']]
Only a few binding correction factors for inside binding have been published, based on rat liver mitochondria or membrane models under very different conditions (temperatures, mitochondrial membrane potential,…) While different mathematical approaches were used to describe the binding an attempt to convert these factors between different mathematical models shows quite similar values for the probe TPMP+ (the probe for which most values are available):
* Brand<ref name="Brand1995"/>: 2-3.2 Β΅l/mg (converted to Rottenberg's system)
* Rottenberg<ref name ="Rottenberg1984"/>: 2.4 to 3.7 Β΅l/mg
* Kamo<ref name="Demura1987"/>: 2.7 Β΅l/mg (converted to Rottenberg's system)
Simultaneous variation of outside and inside binding parameters show:
* the invariance of delta delta Psi
* that strong deviation from published Kin' values do not lead to reasonable results:
[[File:Isolated mito KinandKout variation.png|500px|alt=various delta Psi values and one delta delta Psi values plotted against varying external and internal binding parameter Kout'|various delta Psi values and one delta delta Psi values plotted against varying external = internal binding parameter Kout']]
Conclusions for isolated isolated mitochondria:
* Absolute change of delta Psi values can be obtained.
* Precise absolute delta Psi values can not be obtained without actually measuring the binding correction factor for the studied system. Literature values will usually not be available for the desired system. Approximate delta Psi values may be obtainable by using literature values, if variances in "unspecific binding" betweenΒ  sample types and conditions are small (still to be shown).
=== Permeabilized cells, homogenates, permeabilized fibres and unspecific binding outside the mitochondria ===
In these sample types the determination of mitochondrial protein present is more complicated than for isolated mitochondria. Estimations may be based on the observed O2 flux, or on using a other marker for the presence of mitochondrial activity (citrate synthase). If the amount of mitochochondrial protein was estimated wrongly this may lead do drastically and obviously wrong absolute membrane membrane potentials.
Below the influence of different assumption for the amount of mitochondrial protein in a preparation of brain homogenate. Delta delta Psi values between states of reasonable high membrane potential are not affected.
[[File:Homogenate brain centrifuged Pmt.png|500px|alt=various delta Psi values and one delta delta Psi values plotted against varying amount of mitochondrial protein Pmt'| brain homogenate, centrifuged: various delta Psi values and one delta delta Psi values plotted against varying amount of mitochondrial protein Pmt']]
In these sample types there is a large amount of materials outside the mitochondrial matrix present. But potentially even more difficult than the absolute amount of material is the variety of materials. Inside the mitochondrial matrix the mitochondrial membrane is the only type of material taking up the probe ion and can therefore be accurately described by a single binding correction factor. Outside the mitochondria there may be membranes, proteins, other lipid compartments and even components of the medium to consider. It is reasonable to expect that all of them show a different affinity to TPP+ or other probe ions.
In theory, the four compartment approach can be applied to such samples. All outside material will be exposed to the low extra-mitochondrial probe ion concentration and can therefore be included in compartment D. Due to the different nature of the outside material it can be expected that a quite different binding correction factor will be needed than the one determined by Rottenberg for the outside binding to isolated mitochondria. Additionally, it may be discussed what would be a good marker for the amount of outside material present. It should be remembered that each binding correction factor is only valid for the use with a specific marker quantity (like protein content).
From a mathematical point of few the contribution of outside binding does not cancel even for the determination of change of delta Psi.
However, the first question before addressing this problems is whether outside binding is relevant at all. Brand<ref name="Brand1995"/> stated that for permeabilized cells outside binding may be ignored for high mitochondrial membrane potential. Initially, this seemed to be confirmed by our own initial sensitivity studies. Using outside binding correction factors similar to the inside ones and using protein content as marker, changing the outside binding correction factor by several hundred percent caused comparable small changes in reasonable high membrane potentials and negligible changes in delta delta Psi values for permeabilized cells. However, with growing experience it became evident that unspecific binding may be underestimated by this approach, resulting in obviously too high membrane potentialsΒ  especially for states of known low potential. Part of the unreasonable high membrane potential could be explained by wrong assumptions for the amount of mitochondrial protein (Pmt). Non the less,Β  modeling of the outside binding correction factor showed that sometimes the correction had to be increased by factors above 25 to model reasonable membrane potential. With such a huge contribution of outside binding also differences between states (delta delta Psi) are now very significantly influenced by the choice of the outside binding correction factor. A bit surprisingly, very high membrane potentials still change only very little even when the outside binding correction factor is changed by more than a factor of 25.
[[File:Homogenate brain centrifuged Kout.png|500px|alt=various delta Psi values and one delta delta Psi values plotted against varying external binding parameter Kout'| brain homogenate, centrifuged: various delta Psi values and one delta delta Psi values plotted against varying external binding parameter Kout']]
One problem with this approach, at least in the shown example, may be that medium membrane potential values (e.g. ADP) decrease quite strongly with increasing external binding, resulting in a very strong increase in differences (changes of delta Psi), even if the low potential states are modeled not to a delta Psi of zero but one similar to the values observed for isolated mitochondria.
However, in other but similar experiments medium high membrane potentials (ADP) and changes of delta Psi were more stable against variation of ''K''out'.
[[File:Homogenate brain crude Kout.png|500px|alt=various delta Psi values and one delta delta Psi values plotted against varying external binding parameter Kout'|crude brain homogenate: various delta Psi values and one delta delta Psi values plotted against varying external binding parameter Kout']]
More comparative values both from isolated mitochondria and from homogenate/ permeabilized fibers of the same sample type would be necessary to evaluate this strategy.
The statement that outside binding may be ignored in permeabilized cells for high membrane potentials was actually verified, only with the restriction that this hold only true for the very highest membrane potentials obtainable. This is potentially an important observation for researchers more interested in comparing just one state between different samples. It might even be argued that for very high membrane potentials an absolute delta Psi may be estimated regardless of the used binding parameters. However, this certainly needs further evaluation.
By increasing both the interior and external binding parameters it is again (as with isolated mitochondria)seen that strong deviation from published Kin' values do not lead to reasonable results:
[[File:Homogenate brain centrifuged Koutand Kin.png|500px|alt=various delta Psi values and one delta delta Psi values plotted against varying external binding parameter Kout'|various delta Psi values and one delta delta Psi values plotted against varying external binding parameter Kout']]
There are currently no good methods known to determine the outside binding with the possible exception of radio-tracer experiments similar to those used to determine inside binding. Even if such experiments were done, due the heterogeneity and diversity of materials found in the outside compartment, the results would be less transferable to other sample types than the results for inside binding. An obvious way out would be to use a known state of zero membrane potential to determine either all or at least just the outside binding correction factor. This approach faces two problems:
# It is not clear how a state of reliable zero membrane potential can be reached. The true membrane potential at β€œlow potential” states, like after addition of FCCP, may or may not be zero.
# As discussed above the accuracy of the entire method inherently decreases with decreasing membrane potential. At zero membrane potential the smallest error in measured TPP+ concentration will cause huge errors in delta Psi. In effect the point of lowest accuracy would be used to calibrate the entire method.
However, at least to obtain a plausibility analysis it is certainly helpful to look at this low membrane potential states. A thorough literature search for membrane potentials obtained(with a radio-tracer method) e.g. after treatment with FCCP should be performed. Maybe an solution would be to use literature values obtained for unspecific binding in isolated mitochondria to model the inside binding but use a (crude) approximation of outside binding by observation of a zero mitochondrial membrane potential state.
In summary, there are two obvious ways to obtain binding correction parameters that will allow a more quantitative approach:
* direct determination of outside binding,
* comparison with results obtained for isolated mitochondria under as similar as possible conditions followed by fitting the binding parameters to obtain comparable results for both types of sample preparation.
Both approaches face several theoretical and practical differences, but should be further explored.
=== Further modeling options ===
# Saturated binding: The four compartment model could be extended by further parameters. One possibility would be to allow for a saturable component of "binding". The amount of TPP+ bound would depend only on some proportionality factor and the amount of biological material present but not on the free TPP+ concentration near the compartment. Such a behavior could be detected by performing experiments at different TPP+ levels. To obtain significant differences it would be probable necessary to use very different TPP+ concentrations (Factor 10) resulting in inhibition by TPP+ for the higher concentration studied.Β  This might be solved by using only results at low membrane potentials. However, at low membrane potentials the accuracy of the method is inherently low.
# It was suggested to use the quantity "taken up TPP+ per mass of sample (protein content)" as a relative expression for the membrane potential for given experimental conditions. One advantage is that if the result is to be a relative number anyway, it may be easier to argue (e.g. with reviewers) to use this expression than to calculate some delta Psi and than declaring: "This is not really delta Psi, but some relative value". On the downside, the comparability between different experimental conditions is certainly worse than with some calculated "relative delta Psi, plus even for the same experimental conditions the relationship between the stated number and true membrane potential (especially the linearity of the relationship!) may be worse. This should be checked by calculations / simulations.
While probable not utilizing the measured data to its full extend this approach might be quite a safe way to present some minimum information of the data.
# Methods based on different kinetics of unspecific binding vs mt uptake.
# '''Please add your suggestions!'''
== Correction for substance specific effects on the TPP signal ==
The necessity to perform a TPP chemical background experiment is explained in MiPNet 14.05. Some additional considerations:
=== When to apply a correction ===
For isolated mitochondria absolute delta delta Psi values seem obtainable, see above. Approximate delta Psi values seem to be principally obtainable, though with relying on literature data. The strongly quantitative approach enabled thereby calls for complete quantifications including correction for unspecific effects.
For permeabilized cells, homogenates, and permeabilized fibres, absolute values of delta or delta delta Psi seem currently difficult to obtain. Data will have to presented as a relative value. Therefore, a discussion about to apply or not to apply a correction for substance specific effects seems justified: Whenever changes of mitochondrial membrane potential during an experiment are of interest a correction is most definitely needed. Otherwise even the nature of the change (increase / decrease) may be misjudged.
When membrane potentials obtained by different protocols but using the same parameters (binding correction factors) during calculation should be compared to each other, correction for substance specific effects has to be done, even though only relative values are compared to each other.
When relative values for membrane potentials of the same state obtained via totally identical protocols are to be compared between different samples a correction may not be strictly necessary. In this case the research will have to judge on a case basis. If the correction is obviously rather difficult,Β  the danger of introducing additional errors may be greater than any benefit from getting slightly more realistic values.
Even if it is decided for a particular study not to apply the correction the TPP+ chemical background experiments should be done non the less to detect possible problems.
=== Substances ===
Azide N3- has a very huge substance specific effect. A correction does not seem feasible.
The substance specific effect of ADP is comparable large and should be considered carefully.
== References ==
<references/>
* [[MiPNet14.14 PermeabilizedFibrePreparation]]
* [[Pesta 2012 Methods Mol Biol]]




[[Category:OroboPedia]]
[[Category:OroboPedia]]

Revision as of 18:43, 6 February 2016

Publications in the MiPMap
O2k-Protocols
Ion selective electrode for TPP+ and high-resolution respirometry. Β»Bioblast pdfΒ«

Β» Versions

OROBOROS (2011-12-11) Mitochondr Physiol Network

Abstract: Sumbalova Z, Fasching M, Gnaiger E (2011) Ion selective electrode for TPP+ and high-resolution respirometry. Mitochondr Physiol Network 15.8.

Tetraphenylphosphonium (TPP+) accumulates in the mitochondrial matrix as a function of the mitochondrial membrane potential. The TPP+ electrode is an ion selective electrode (ISE). The voltage signal [V] is linearly dependent on the logarithm of the free concentration [TPP+].

Β» Poster
Β» O2k-Protocols
Β» Product: Oxygraph-2k, ISE, O2k-Catalogue

β€’ Keywords: O2k-MultiSensor System

β€’ O2k-Network Lab: AT_Innsbruck_OROBOROS


Labels: MiParea: Respiration, Instruments;methods 





HRR: Oxygraph-2k, TPP, O2k-Protocol 

O2k-Demo, O2k-MultiSensor