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| The examples in the Membrane Potential protocol [http://www.oroboros.at/index.php?id=protocosl_tpp-membranepotential [MiPNet14.05]] are with permeabilized cells not permeabilized fibers. In the meantime, we have also worked with isolated mitochochondria - as expected this proofed to be easier than the permeabilized cells. Indeed that is the reason we started with permeabilized cells: Early experiments showed it doable but since its more difficult than isolated mitos all performance results obtained should be transferable to (or even be better with) isolated mitos. But what about permeabilized fibers?
| | #REDIRECT [[Mitochondrial_membrane_potential#Mitochondrial_membrane_potential_of_permeabilized_fibres]] |
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| == General ==
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| As for permeabilized fibers one can adopt two different positions:
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| a.) we have not found any reference that describes this to have been done. So one could see it as a totally new, presumable difficult technique, that needs a lot of methods development.
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| b.) on the other hand, one can argue: whatβs really the difference between permeabilized cultured cells and permeabilized fibers. Maybe a bit more of non mitochondrial cell material, but otherwise?
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| We certainly plan to extend (if possible) this technique to permeabilized fibers. The first step definitely is to guess the required sample amount. From experiments with isolated mitos or permeabilized cells one can see what concentration of mitos is necessary to obtain noticeable differences in the TPP concentration. This will have to be converter (e.g. via the respiratory rate) to a amount of sample necessary for permeabilized fibers. Maybe even more sample is necessary to compensate for more "outside binding" in permeabilized fibers. Even with permeabilized cells higher sample amounts are required, as compared with standard high resolution respiratory measurements. Then there have to be some modifications in the protocol, especially how the sample is introduced. It is important that the total amount of TPP in the chamber is known at all times. It follows that the sample may not be preconditioned outside of the chamber to TPP, and even a rough estimation of the sample volume will be necessary.
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| If we go now totally into the realm of speculation: Might there be any specific reasons why the method just does not work with permeabilized fibers even after method development?
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| One could postulate that rather big junks of very hydrophobic material (fat) might "hover" up all the TPP. However, due to the far higher TPP concentration inside the mitochochondria as compared to the outside concentration (because of the membrane potential) external "unspecific binding" is usually nearly negligible for permeabilized cells......
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| == Introduction of the Sample ==
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| The established was 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 indented measuring chamber. With the final calibration step the planned starting TPP concentration is reached. Then the sample is injected into the "calibrated" chamber. Therefore, unlike in the application of other potentiometric methods (pH, Ca2+,..) the "calibration" does in fact serve TWO different purposes:
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| 1.) calibration of the sensor (of course)
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| 2.) establishing the total amount of TPP present in the chamber. This amount has to be a precisely known for the calculation of delta Psi from the measured [TPP]
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| ===Problems===
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| Introduction of the sample is a key problem in extending the TPP+ method for measuring mitochondrial membrane potential for two different reasons:
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| 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, ...)
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| 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:
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| 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:
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| 1.) any mixing of the solutions before the displacement is complete
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| 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)
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| All methods to introduce a sample have to consider both problems.
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| === Possible Methods===
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| 1.) The obvious method: '''opening the chambers''' and placing the permeabilized fiber in the chamber:
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| 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 βbubble fightingβ 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.
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| 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:
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| * End of calibration run, top of stopper is dry
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| * Plug for βsample portβ is removed: since top of stopper is dry no additional liquid gets into the chamber
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| * 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)
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| * 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 βsampleβ 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.
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| 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 βsample portβ 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.
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| 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.
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| Advantage: No modification of stopper required, same development of βintroduction skillsβ necessary as for method 2. Same advantages / disadvantages in regard to βProblem Bβ as for Method 2
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| 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.
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| 4.) Introduction of the sample via the '''titration port''': Requires obviously the most sophisticated tool for getting the sample in, but only minimal distortion. ?steel wire?
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| 5.) Using a '''partially homogenized sample''', injecting it: probable not doable with current (and possible) permeabilization protocols.
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| It may be that with increasing practical skills even method 1 is doable, but otherwise a method has to be found to insert the sample via a relatively narrow bore:
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| Biopsy needle ?
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| Steel wire?
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| Question to permeabilization experts: What tool do you need to get a permeabilized fiber through either a 2.6 mm or an 6.2 mm port (length 50 mm)?
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| === Evaluation and SolutionΒ by the Neufer Group at East Carolina University===
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| The group of Darrell Neufer at East Carolina University, Greenville, NC, USA, a [http://www.oroboros.at/index.php?id=1312 MiPNet Reference Lab] evaluated two of the discussed approaches and presented a solution. For their full contribution see the Discussion page. In summary
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| * the port for the reference electrode is used to introduce the sample
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| * the sample is split into several parts
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| * if necessary, the sample pieces are introduced into the chamber using a standard Hamilton syringe and the reference electrode itself
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| * other methods for sample insertion were tested and rejected
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| '''Until full integration of their method into this page see the contribution of the Neufer group on the [[Talk:Mitochondrial_membrane_potential_of_permeabilized_fibers|Discussion page]] for a detailed description.'''
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| * also a method for gas phase re-oxygenation was tested by the Neufer group. With this modified gas phase method distortions could be kept to a minimum. It should be noted 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 ( ΞcO2 β€200 ΞΌmol/l) this seems to be an alternative.
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| {{#set:Scientific service=Membrane potential|Scientific service=Permeabilized fibers}}
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| __SHOWFACTBOX__
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| {{Troubleshooting}}
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