Talk:MiPNet19.03 O2k-cleaning and ISS

Contamination with hydrophobic inhibitors: The experiences with TPP electrodes

• We had problems with inhibitors carry over during the experiments with TPP electrodes. Despite of all the standard cleaning procedure of the chamber and electrodes, the inhibitors were present in next experiment.

• We rinse the electrodes after use with water, dry with paper, rinse with absolute ethanol, again dry with paper and rinse with water again. This procedure is sometimes not sufficient.

So we decided to put the electrodes rinsed with this procedure into a falcon tube filled with water for a while (15-30 min). After the standard washing of the chamber with water, 70% ethanol, absolute ethanol and water we did washing of the chamber and electrodes (in the chamber) with liver homogenate. It really helped to get rid of all the inhibitors in 90% of cases.

We are very careful with using syringes and cleaning them after each titration if the inhibitor was present already in the chamber (wash them with water by putting them in falcon tube with water drying with paper and putting to ethanol and drying with paper again). For each chemical we have a separate syringe. We wash it after the experiment and store filled with absolute ethanol and with the tip in absolute ethanol (in falcon tube). Also each TPP and reference electrode has its own falcon tube with storage solution.

Zuzana 11:08, 9 December 2010 (CET)

Apparent carry-over of CI-inhibitors versus contamination in chemicals (update: solved)

Partially unresolved

We have been trying your suggestion about using liver homogenates to check the problem of our Oxygraph-2k. I am happy to see that your suggestion works, but not completely, as you can see in files attached. You will find files from two different O2k instruments. In summary, first day after letting liver homogenates to incubate with PM we did not see any response when we have added ADP. We washed and let it stay for few hours with a new liver homogenate and the good news is that one of the chambers responded to ADP addition after PM (first time since I am here that I see this response in liver). So we decided to continue with the treatment with both Oxygraph and we got 3 of the 4 POS responding to ADP after PM in liver homogenates. Two days ago we have tried then to test hypothalamus to see if we are able to see the responses I use to see in these homogenates and you can take a look to the file “2012-02-15 Hyp-liv” we saw an small effect of ADP after PM but far from the one I use to see (the addition of Succinate as always works well). In general, I think we are in the right track but it needs a more strong washing of the carry-over effects of rotenone or other inhibitors (I started to think after analyzing many files that this problem is being present for a long time and that the concentrations of the inhibitors are above recommended even they were prepared several times) This high concentration of the inhibitors could explain that after incubating with cells for several hours still we come back to the same problems for continuing using the same titrations of the inhibitors. However I am not so sure about it and I will test the concentration once we solve the problem and we can be sure that we do not have more carry-over effects. Do you have any suggestion of how can I get read of the carry-over effect? Once we solve this we could check if we have solved finally all our problems and we can start to work in our projects using the PBI-Shredder.

Pablo

How to continue

The problem of washing/carry-over of inhibitors is an extremely important issue. In our projects (practically always involving 4 to 6 O2k-Instruments with 8 to 12 chambers in parallel) we have had phases (largely unexplained) where such problems became apparent, and then (partially unexplained) they disappeared.

Consider that you would not have the experience of your experimental phase at Karolinska. Probably, either, you would not recognize the methodological problem and run into a series of unnoticed artefacts, or you would put into question the entire approach. We have not encountered the latter scenario - according to all the feedback received or not received from more than 350 labs . But I am afraid, that there is a grey zone of labs that use problematic results uncritically (without having the necessary experience). Hence the problems your are facing (and which hopefully will be resolved soon) should be communicated to the O2k-Network: Perhaps the corresponding feedback may help us to solving the problem more quickly. Or perhaps, at this stage it may merely be an educational pathway for sharing the experience with others. Therefore, we should aim at providing a summary of this – hopefully successful – trouble shooting, in the spirit of QA and Gentle Science. At OROBOROS INSTRUMENTS, I want to implement the strategy of OPEN INNOVATION. Open trouble shooting has to be part of it.

Now some considerations coming back to the specific problem. We are not alone with this problem. Some groups using conventional oxygraphs (with Perspex chambers, Teflon stirrers etc) have long avoided some carry-over effects by strictly separating chambers with application of rotenone from chambers without. This, however, is not satisfactory for us, since it precluded application of our complex SUIT protocols. But if the apparent carry-over problem is only partially resolved, it would make sense to try a series of very simple experiments, when one O2k-Chamber is reserved for CI-linked respiration, without further addition of a CI-inhibitor. Still you can add succinate after CI-linked substrates. A series of such runs would reveal higher CI-linked OXPHOS capacities compared to chambers where CI-inhibitors are regularly used. This would not represent a finally satisfactory state, but could provide a next step for diagnosis of the underlying problem. Perhaps we may find some new-generation strong glass-ware detergents or other stuff, which destroy all these inhibitors but can be washed out more easily (so far, I stayed away from detergents other than saponin and digitonin). Perhpas it might be even worthwhile to try digitonin as a washing reagent (???).

I a series of experiments with the TPP+ ion selective electrode, we encountered a washout problem that increased over several months. Originally some outliers were observed occasionally. And within several months, the problem become more dramatic, with more than half the chambers remaining suspect. At the same time, no carry-over effects were apparent in the same chambers, when no MultiSensor experiments were carried out. The phenomenon remained mysterious, and our PostDoc project was finished without resolving this crazy issue. Other groups using our TPP+ electrode did not run into such an extreme problem (so far?). Our current projects, and the TPP+ demos at our workshops, did not reveal this carry-over problem. But it may start again tomorrow!?!

I hope by combining the critical observations from various experimental settings, we may shed some light on these problems, hopefully developing a general solution.

Many thanks for your patience, open discussion, and good luck to all of us in finding a lasting solution.

--Gnaiger Erich 21:31, 17 February 2012 (CET)

Testing the chemicals

Today we have performed another experiment looking for the solution of our Oxygraph-respirometry problems. In order to be sure that what we are facing is a problem of carry-over of inhibitors due to an excess in the titrations or a defect during the washing period, we decided to perform another experiment. This time the idea was to test that all our substrates and media that are involved in the non existing response of Complex I substrates to ADP addition (M, P, G, ADP, MgCl2, and MiR05) were tested in a different Oxygraph-2k from another group, which does not experience any problems with inhibitors. If our experiment works well in their O2k, then we will be more certain that the problem is a carry-over effect of the inhibitors in our O2k-chambers. However, the results of this experiment tell us that this is not the case, because we got the same problems (lack of response to ADP in liver and hypothalamus homogenates in MiR05 with M+P+G) - same results as experienced in our O2ks. Then, if we exclude a carry-over effect of the inhibitors as the source of our problems (I am not 100% sure about it yet), what could be the reason for this lack of response to ADP?. Again, as always, addition of succinate after M+P+G+ADP increases respiration dramatically. - Pablo 2012-02-22

I agree, you are now zooming in on the quality control of the chemicals, in contrast to a potential carry-over of inhibitors from the O2k-chamber. You tested the medium MiR05 before, without indication that this is the source of inhibition. Then the added chemicals have to be tested. In an early phase of bioenergetics, ADP was noted to be contaminated by zink. Zn2+ is an inhibitor of CI-linked respiration and less potent for CII-linked resipration (Brown et al 2000 PMID: 10788456; Ye et al 2001 PMID: 11226237). The best test would be to repeat your experiment using all chemicals from the other lab in your O2k. If you would get now the expected ADP-stimulated OXPHOS capacity, then you could immediately titrate you own ADP, followed by your M, P and G. Perhaps you would observe an inhibition after one of these titrations. Then the problem would not be specifically related to the 'hydrophobic inhibitors'. Quality control of chemicals (applied in our media and SUIT protocols) is a very general emerging problem, which requires new strategies to be solved. - --Gnaiger Erich 11:34, 22 February 2012 (CET)

Problem solved

The problem was finally solved. Some of the ethanol used in Pablos lab contained small quantities of an antiseptic. See Contamination_by_hydrophobic_inhibitors#Unintentional_introduction_of_inhibitors_to_the_O2_chamber

Cleaning with diluted acids

Question: Colleagues and I want to introduce heavy metals into the chambers of the oxygraph-2k. We were wondering what that would mean in terms of cleaning: Would acid-washing the chambers with either 0,1-0,3N HNO3 or HCl be a problem for the membranes?

Answer: The glass chambers and membranes (FEP) would not be a problem, but some polymeric components might. Therefore, we tested approx 0.3 M HNO3 over night at room temperature plus 2 hours at approx 35 °C on the following materials: FEP (POS membranes), PVDF ( white stoppers), POM O2k-Chamber Holder), butyl rubber ( OroboPOS-Seal Tip, and Viton O-ring\Viton\12x1 mm. No immediately visible problem was detected. Frequent or prolonged use might of course still have negative effects. Since HNO3 was nor problem for the polymers (non oxidizing), HCl of similar concentration is presumably save.

Of course 0.3 M HNO2 is still a rather mild way to deal with heavy metal contaminations. Classical methods (cocentrated acids) would require to remove the glass chamber and clean it outside the O2k as described for protein contamination MiPNet19.03_O2k-cleaning_and_ISS#General_cleaning_instructions. Fasching Mario 15:45, 20 August 2014 (CEST)