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Talk:MiPNet15.08 TPP electrode

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Introduction of the Sample into the Chamber

Evaluation of Methods and Solution Presented by the Neufer group

This contribution is based on a report by Lin Chien-Te (Peter) from Darrell P. Neufer's group at

Department of Physiology
Brody School of Medicine
East Carolina University
Greenville, NC  27858, USA


Because the Neufer group does not (yet?) have a user account on this wiki, this contribution was placed, formated, and edited by Mario with the permission of Prof. D. Neufer. Due to the necessary editing any spelling errors, un-justifiable omissions, etc. are entirely the fault of Mario.

Background:

  • Previous experiments found that opening up the stopper will drop the TPP signal significantly.

Question:

  • How to introduce permeabilized SKM fiber without to affect the TPP signal significantly?
  • How to do re-oxygenation without to affect the TPP signal significantly?


Conclusion:

  • Introduction of sample

Never allow the TPP electrode to be exposed to air or an air bubble during your experiment. It will decrease the TPP signal significantly. Opening up the chamber (stopper) is even worse. The reference electrode can be exposed to air without a big impact on TPP signal. Use the reference electrode port to introduce the fiber:

  1. Blot-dry the fiber before getting it into the chamber
  2. If the muscle is too big for the reference electrode port, split it into 2 or 3 pieces, so that it is easier to get through the port.
  3. Switch stirring off before withdrawing the reference electrode.
  4. Use a Hamilton syringe needle (25ul) (or other tool) to put the muscle deep into the hole. Use the reference electrode to push the muscle in slowly and gently so that no damage can be caused to the muscle fiber and no buffer can come out from other ports.
  5. Move up and down the reference electrode a little (and TPP electrode if necessary) to get ride of the air bubble in the chamber, if it exist. Make sure no air bubble contact the TPP electrode tip during this process.
  6. Switch stirring on.
  • Re-oxygenation

Should try to start the experiment with higher [O2] to prevent re-oxygenation. Re-oxygenation without any air bubble contact of the TPP electrode tip cause only little, but not 0, effect on TPP signal.

  1. Stop the stir bar
  2. Lift up the stopper and allow a small air bubble in the chamber but not too big to contact the TPP electrode tip.
  3. Inject O2 into the air bubble. Caution: cannot inject into the buffer phase because it will cause some more bubble and some buffer may escape from the chamber.
  4. Stir bar on to reach the desired [O2].
  5. Stir bar off and drop the stopper slowly to get ride off the air bubble. If necessary, move up and down the electrode to help.
  6. Stir bar on.

Others.

  • When use permeabilized fiber, the total [TPP] in the chamber and the tissue weight (and [O2]) have to be optimized in order to get a sensitive measurement.

further results (summarized by Mario):

  • when the TPP electrode was removed and reinserted this always led to bubble formation and a huge change in TPP signal
  • when the sample was too big and not split up it, it formed a ball when trying to push it in with the reference electrode and blocked the port

Comments to the Contribution from the Neufer Group

First, I would like to thank Peter and the entire group of D. Neufer for the great work the did in developing this method and especially for their willingness to share their experience.

  • I think we can conclude that introduction of the sample seems at the moment to be the best method and that it has at least the potential to solve the problem entirely.
  • Unfortunately, any unintended change in the chambers total TPP content shows itself only in its full effect when doing the final calculation of the mitochondrial membrane potential. Therefore, still some fine tuning might be necessary to either totally prevent any change or to be able to calculate such a change.
  • Is it possible to estimate to volume of the fiber sample? The same volume of TPP containing medium would be replaced by the sample, enabling calculation of TPP loss.
  • As for re-oxygenation: The easiest was would be to use the H2O2 injection / catalase in the medium approach see MiPNet14.13, keeping in mind the limitation to a delta c(O2) of 200 ยตM. This would avoid opening the chambers for re-oxygenation altogether. Is there a special reason you want to avoid the catalase? Also, using H2O2 / catalase does not require to have the chamber at high O2 before introduction of the sample, for increasing the oxygen levels by a delta of 200 ยตM above air saturation.If even higher oxygen levels are required introduction of a high oxygen level by the gas phase method may be combined with re-oxygenations by peroxide injections. First introduce the sample, then achieve the desired final oxygen level by injection.
  • Maybe be could find something else than the reference electrode to push the sample further down (the mounting tool for the ISE?). I am little bit worried about the survival of the reference electrode....

Mario

Description of the experiment with permeabilized fibres from mouse heart

  • We calibrated the electrode at high O2 level >500 nmol/ml up to 1.5 ยตM [TPP+].
  • After calibration we lifted the stopper with electrodes just slightly (stirrer off) (the TPP electrode was still in the liquid). We removed the reference electrode and introduced fibers through reference electrode hole using glass Pasteur pipette cutted to the length 16.2 cm. The cutted edge was smoothed by the use of fire.

The fibres were soaked into the pipette and gently introduced into the chamber. We used around 3 mg of fibers per chamber.

  • Then we closed the chamber without bubbles and switched the stirrers on.

This worked nicely and we got good traces of TPP signal. The O2 flux was quite high and needed frequent reoxygenation. In future we will try to use less tissue and go to 1 ยตM [TPP+].

Zuzana 10:36, 9 December 2010 (CET)


Correction for TPP chemical background

Question: I use the "internal" chemical background correction mainly because of the reason of experiment to experiment difference. More importantly, the permeabilized skeletal muscle fiber TPP releasing or uptaking is always much slower than the isolated mito based on my experiments. When use fibers, a sudden change in TPP signal mostly is not due to the sudden quick uptake or release but due to the background chemical affection. Therefore, I choose the TPP signal right before and after a chemical addition in the presence of FIBERS for the background correction. Personally, I feel it is more accurate because we can avoid the experiment to experiment difference issue (an experiment with biological tissue vs tissue free experiment). The chemical background effect may not the same in different [TPP] condition. A biological tissue contained experiment should not have the same [TPP] in the bath when compare with tissue free experiment, chemical background effect experiment. That is what I think. I would like to hear how you experts think. Any feedback will be greatly appreciated!!!

Answer: Just to clarify: Do you do (A) a dedicated background run, just in the presence of sample in a state were you assume no changes in membrane potential to take place because you actually killed the cells already before (an approach also Zuzana is sometimes exploring) or do you (B) just assume there is no quick change in membrane potential at all and use the injection form the real biological experiment to do this correction? The chemical specific responses at different TPP concentration are not so much different but the sample might take up a considerable fraction of some of the used chemicals and this would be a reason for a determination in the presence of sample (we do not know). However, in my opinion this would only be possible if you are sure no change in membrane potential is happening at all. I do not have your experience with TPP and permeabilized cells but I would not dare to speculate that the changes in membrane potentials are sufficiently slow for method B. We do everything do keep the relative size of the correction small as compared to the real change in membrane potential. So if the correction is 10% or the real change, and lets say just 10% of the real membrane potential change happen immediately this would introduce an error for the correction of 100%. What size (relative to the real chance) to your corrections have? Zuzana your opinion? Mario 09:50, 3 February 2011 (CET)


Reply to Mario: (by Peter Lin, Neufer's group) close to (B) but not totally. I assume there is a quick change in mito membrane potential, however it take more time for the [TPP] between the assay bath and mitochondria matrix to reach equilibrium due to the structure nature of permeabilized skeletal muscle fiber. This should be particularly true in a protocol when one try to change the respiration/membrane potential gradually and slowly in conditions such as fine scale ADP titration on top of the substrates supported maximal state 4 respiration. In isolated mitochondria we can see the TPP signal changed and stabilized very fast. In fibers, it take about 25-35 mintues to reach steady TPP signal (voltage) in maximal state 4 condition. It also take at least 4-8 minutes to reach a steady TPP signal when perform a fine scale ADP titration on top of the substrates supported maximal state 4 respiration. The FCCP addition should ideally release the 99% of the TPP in seconds, it actually take more than 5 minutes to reach a steady TPP signal. Based on the observation mentioned above, I assume a quick (~10sec) change in TPP signal after an chemical injection (not the TPP signal spike right during the addition process) is not due to the mitochondria TPP uptake or release but the chemical specific effect on the TPP signal. Any feedback will be greatly appreciated!

Peter Lin


Reply: You are very probable correct in the clarification of the slow mechanism we are discussing here: It is the re-distribution of TPP that might be slow, not (as I implied above) the change of the membrane potential. Maybe this process works actually in a more linear manner (time wise), than a change in membrane potential for which I would always suspect some kind of non linear behavior with a lot of the changes happening early. The long time scales you mention are certainly impressive (unfortunately, and TPP is supposed to be faster equilibrating than TPMP !). So this would imply that this method could be valid. In think when you do this slow titrations you are already approximating the kind of experiment suggested above: Having biological material present but there should be no more change in membrane potential. Non the less it would be very interesting to compare the correction values obtained by this three methods (internal correction, assuming slow TPP equilibration, dedicated experiment but with biological material present in a state where no change in membrane potential is possible, external correction). The second method should have all the theoretical advantages of the first but just with even less danger of extraneously including any "real" changes. But of course it takes nearly twice the time! For most substrates I am rather confident that there will be not to much difference in your case (with this slow changes) anyway. As for the original method (no biological sample present), most substrate specific effects seem not to be depending on the exact TPP concentration and even if there were a dependence this could be taken care of by choosing a proper TPP con in the TPP chemical background. I can not see the sample hovering up all ADP, ethanol, or DMSO from the solution. I CAN imagine happening this for hydrophobic inhibitors, but then we know that those do not have a substance specific effect on their own in the used concentrations (only their carrier solvents). But yes, there could be substances (maybe not in use now) that do have a strong effect on the signal and are significantly removed from the solution by the sample. In such a case the approach of doing the TPP chemical background effect will lead to erroneous high corrections.

Doing things in triplicate is time consuming but I would suggest to compare at least two of the tree methods in at least one experiment before relying on a background recorded in the presence of sample. Thanks again for pushing this development and sharing your thought!

Mario 23:00, 3 February 2011 (CET)


Pmt, PC from literature?

Question: (1) With the TPP spreadsheets, Pmt and Pc values are the values that we use literature values for (that actually measured membrane content), and not actually measured protein (mg/mL) off the sample that is being run? and Vmt I think we also used published values for?

Answer: No, Pmt and Pc describe how much sample you put into your chamber and therefore can not be taken from literature. If you use twice the amount of sample these values will have to double. For isolated mitochondria Pmt = Pc. Please see also the Rottenberg paper cited.

Vmt can be taken from literature, especially as the TPP method is very insensitive against errors in Vmt. Similarly we take Kin and Kout from literature. Vmt, Kin , and Kout are all SPECIFIC values, that is values per amount of mitochondrial / cellular protein. But to use these specific values you need the absolute values of mitochondrial cellular protein- ideally from an assay or at least some rough estimation from the respiration itself (if you know the typical respiration of your sample per mg mitochondrial protein). In the sheet "external input" you see a very rough template for this respiration approach, but a proper assay is of course the way to go.

Some background information :-) : Actually the choice of mitochondrial protein as the marker the specific values Kin and Kout is a bit arbitrary / counter intuitive: The unspecific binding, described by Kin and Kout, actually will take place mainly in the mitochondrial lipids not in the proteins. But since mitochondrial protein is easy to measure and the ratio lipid to protein is hopefully constant all the initial developers of the method used mitochondrial protein as a marker for the amount of mitochondria. For samples beyond isolated mitochondria (permeabilized cells,..) the assumption of a constant lipid to protein ration of course brakes down, maybe one reason why there are no good values for Kout in "cellular samples". Fasching Mario 09:41, 17 April 2015 (CEST)