Talk:Mitochondrial membrane potential of permeabilized fibres

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Mario

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

a MiPNet Reference Lab

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. 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. First introduce the sample, then achieve the desired 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 µM [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 µM [TPP+].

Zuzana 10:36, 9 December 2010 (CET)