Mitochondrial membrane potential of permeabilized fibres
The examples in the Membrane Potential protocol [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 permeabilezed celles: 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. As for permeabilized fibers one can adopt two different position:
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.
b.) on the other hand, one can argue : what’s really the difference between permeabilized cultured cells and permeabilized fibers. Maybe a bit more of non mitochondrial cell material, but otherwise?
We certainly plan to extend (if possible) this technique to permeabilzed fibers. The first step definitely is to guess the required sample amount. From experiments with isolated mitos or permeabilzed 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 permeabilzed fibers. Even with permeabilzed 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. If we go now totaly into the realm of speculation: Might there be any specific reasons why the method just does not work with permeabilzed fibers even after method development? One could postulate that rather big junks of very hydrophobic material (fat) might "hoover" 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 permeabilzed cells......
<ul><li>"TPP" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property.</li> <!--br--><li>"membrane potential" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property.</li> <!--br--><li>"permeabilized fibres" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property.</li></ul>
