Talk:MiPNet15.03 O2k-MultiSensor-ISE

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Carry-over of inhibitors with the ISE

  • I don't seem able to get rid of all the inhibitors after an experiment. After the standard cleaning procedure, I let the electrodes stand in distilled water for about 20 min and then I put them into a homogenate for about 20 min as well. Still, in the next experiment, respiration is inhibited. When removing the electrodes, rinsing the cells and doing standard measurement (without TPP electrodes inserted), respiration is just fine. The only time I get a good measurement is after I replaced the membranes of the TPP-electrodes. This is impossible to do for every experiment.
  1. If cleaning with biological material does not work, the third option of cleaning is to immerse the electrodes in ethanol. This will however, decrease the lifetime of the electrode. Before, I recommend to put the homogenate into the O2k chambers, activate stirring and keep the temperature at 37 °C for this homogenate washing procedure.
  2. I had the same problem with inhibitors - sometimes I could not get rid of it by the procedure you just explained. Maybe the most effective was inserting electrodes into the oxygraph chamber with homogenate like in experiment. You have continuous stirring and 37°C... But even this did not give 100% success.
  • Our recent experience shows that soaking the TPP reference probe in ethanol over time can damage the white part (ceramic) in the tip and cause the TPP signal not to steady. To prevent it, it is a good idea to have the reference probe (or just the glass part) designate to each inhibitor condition so that there is no need of using ethanol to wash the reference probe. US NC Greenville Neufer PD

Increased instrumental O2 background

  • I suspect biological contamination of one of my TPP electrodes. Oxygen flux in the closed chamber, without biological material, is too high (~10 pmol/(s*ml)). This isn't the case when measuring without the TPP electrodes. So, I'm quite sure that the contamination must come from the TPP or reference electrode. How can I get rid of this? I didn't want to take the risk to put the electrodes in 70% EtOH because I'm not sure if this would be OK.
  1. You can rinse and immerse the TPP and reference electrodes in ethanol, but this will degrade membrane lifetime. But since the other option is to disassemble the electrode, you should try the immersion in 70% ethanol. If this does not help and you have to disassemble the electrode: wash all the individual components of the electrode first with water and then with 100% or 70% ethanol for prolonged times. If you saw biological contamination for a TPP electrode, remember to also replace the storage solution of the electrode.

For the reference electrode the problem with long exposure to ethanol is potential clogging of the fritt. If you suspect biological contamination I would just exchange the glass barrel: Use a new glass barrel for the time being. In the meanwhile clean the old barrel first with water (soak it in water to ged rid of the salts), then immerse it in 70% ethanol as long as you want to. Before refilling the glass barrel clean it again with a lot of water to get rid of all the ethanol. ~ Fasching Mario 13:24, 3 May 2012 (CEST)

  1. Contamination of the TPP electrode can also be a problem. It is necessary time to time exchange the storage solution and keep electrodes in the darkness. I could see several times something growing inside the storage solution, but never had such a high O2 flux after good rinsing and keeping in fresh solution. You can easily clean the reference electrode by removing the liquid, cleaning the glass part and putting it into ethanol. It is easy and fast. For cleaning the TPP electrode you would need to remove the membrane, clean everything with ethanol, condition the electrode... it would take 3 days. ~ user:Sumbalova Zuzana
  2. So - maybe the only solution for standard experiments would be to choose a different approach - choose some particular problem to solve: the effect of stepwise addition of ATP on membrane potential, the effect of Ca2+...on mitochondria of control and affected animals... Maybe malonic acid does not stick to the membrane of TPP electrode - it is successful used in the literature, see this paper:
Borutaite V, Morkuniene R, Budriunaite A, Krasauskaite D, Ryselis S, Toleikis

A, Brown GC. Kinetic analysis of changes in activity of heart mitochondrial oxidative phosphorylation system induced by ischemia. J Mol Cell Cardiol. 1996 Oct;28(10):2195-201. PubMed PMID: 8930814.

  1. I know from Vilma, that they keep the electrode contaminated with rotenone only for measurements with rotenone. And they have long-lasting experience with home-made TPP electrodes. So - my advice is to find a good application for TPP electrode from the literature, where you don´t need to use lipid-soluble inhibitors. ~ user:Sumbalova Zuzana