Talk:Oligomycin

MiPNet discussion forum: problem with Omy (2013-10-22)

Steffi Wohlgemuth

The problem concerns the unresponsiveness of our cells, isolated mitos, and permeabilized muscle cells to oligomycin. When we titrate oligomycin to our cells or to our permeabilized muscle fibers (and the same happened with isolated mitos in the past in another lab) to induce state 4 or leak respiration, the flux only decreases by a little bit, sometimes returns to the flux before oligomycin-addition after a short while. -- I have abandoned the O2k for a few months and want to pick up those experiments again, but my students are reluctant to run the oligomycin-induced state because it has almost never worked and prolongs the protocol with no added information. -- I am sure I will be asked what concentration of Oligo we added: The recommended concentration of 2 ug/mL of a 4mg/1mL EtOH stock. We even started making a little bit more dilute Oligo stock in order to titrate smaller amounts up to the final concentration to see if less oligo would do the trick, if we added to much, and if our cells were more sensitive and less oligo would be required. -- No satisfying results. So my overall question: what is the deal with Oligo? I have heard from others as well that oligo is not quite working as it is supposed to. What can we do differently? Are we doing anything wrong? Is there a better oligo besides the one recommended from Sigma.... Sometimes the batches are different or the manufacturer changes procedures..... Any help or suggestion at this point would be awesome and very, very welcomed. [[email protected]]

Felix Mark

we never had fundamental issues with oligomycin, but in our assays with temperate and polar fish and cephalopod permeabilized fibres (heart muscle), it sometimes takes ages for a result to become visible and stable. So it can depend on assay temperature and time, as well. At low temperatures, eg. 0-5°C, we sometimes wait up to 45min. Have you tried atractyloside? It's not quite the same response as it blocks the ANT rather than ATPase, but should give similar results in terms of flux reduction and membrane potential. And, at least in our preps, it's much faster. We sometimes use it to double-check. I assume you have also tried preparing a new stock solution from a fresh new batch of oligomycin, that's usually our last resort…. [[email protected]]

Marten Wikstrom

I would recommend the following:

First test whether your oligomycin will block the ADP (+ Pi)-induced increase in respiratory rate (State 4-->State 3) with isolated mitochondria respiring on succinate. That will test whether your oligo batch is working/whether you use a sufficient concentration of oligo.

If the test is positive (oligomycin blocks the State 4->State 3 transition), then to understand the unresponsiveness to oligo in other experiments would require detailed knowledge of the conditions in those expts.

If the test is negative, throw your oligo batch away and get a new one. [[email protected]]

Senyilmaz, Deniz

What is the brand of your oligomycin? Initially I had the same problem exactly, but then I switched to Sigma, and oligomycin started to work! For some reason, the other brand failed to induce state 4 respiration even though it was able to activate other downstream processes in the cultured cells. [[email protected]]

Lars Eide

Must be your oligomycin batch: we actually experience the opposite problem: oligomycin is hard to get rid of, and extensive wash (of an 7-years old oxygraph) is required to reset the system for next run. [[email protected]]

Christopher Hedges

I have worked with oligomycin in both the Stepto lab in Melbourne and the Hickey lab in Auckland. In Melbourne we have had extensive trouble with uncoupling following oligo (our uncoupled respiration was frequently less than oxphos in permeabilised muscle preps). I haven't personally experienced what you describe with respiration returning to prior levels but certainly had trouble with oligo - ultimately our solution was to stop using oligo and use atractyloside or carboxyatractyloside instead. This may or may not suit what you need to do. An important consideration is that ANT conducts some protons so contributes to leak respiration. We have found that adding atractyloside after oligomycin causes another small decrease in oxygen consumption, even though they theoretically both induce the same state (high substrate, low ADP turnover). I have not used permeabilised cells and my limited work with isolated mito's we had already given up on oligo so this was all found with permeabilised skeletal muscle fibers. [[email protected]]

Michael Duchen

Oligomycin can be terribly 'sticky' - I have had the experience years ago of using imaging dishes (for which the coverslip on which cells were grown formed the base) which had been used for oligomycin previously and the cells behaved as though they had already seen oligo. As it is effectively irreversible is it conceivable that you don't see responses because the cells have effectively already been exposed? It is necessary to wash components very carefully with ethanol.

To test whether oligo has been effective or not, the easiest thing to do is to measure the potential and see how the potential responds to inhibition of respiration using rotenone or cyanide. Under control conditions, depolarisation is rather slow in most cells, but is much faster and more complete after exposure to oligomycin as the ATPase can't run in reverse to maintain potential.

I can't think of anything else other than try a new batch of oligomycin! [[email protected]]

Alessandro Pontoglio

my name is Alessandro Pontoglio, it's three years since I worked and graduated as "Dottore in ricerca" (Doctorate, Ph.D) in the team of Prof. Scatena Roberto, at Policlinico A. Gemelli in Rome, and what I managed was the O2K techincs and technologies applied to hepatocarcinoma cells (HepG2) cultures permeabilized and non permeabilized. Sometimes promielocitic leukemia cells (HL60) cultures.

As I've read in your message : your team is experiencing some troubles with oligomycin flux determination.

I've read you're using muscle cells permeabilized and no permeabilized, and even mitochondria from the same kind of cells. Am I right?

Would you please answer to these questions? It would be worthy in order to try to understand what can be improved or changed. Thank you.

NO PERMEABILIZED CELLS :

1. What are the concentration in O2K chamber, the origin, the age, the number of changes of these cells (if cultured for a long time)?
2. What's the basal flux respiration? Do you think it is already low at the beginning of the determination or it is normal if compared to what is written in the literature?
3. Is the experimental pathway of your experiments : basal-oligomicym-FCCP-Rotenone-Antimycin? Or else? If you think you can disclose them, would you show me the values of the corresponding respiratory fluxes?

PERMEABILIZED CELLS :

1. What's the cell concentration in the O2K chambers?
2. The experimental pathway your team performs is : basal-(malate+glutammate)-digitonin or saponin-ADP-Succinate-ADP-ADP-oligomycin-FCCP-Rotenone-Malonate-Antimycin or another?
3. If any, have you ever seen well shaped spikes when ADP is added, e.g. with succinate?

GENERAL : Supposing the concentration of oligomycin your team started with was the right one :

1. Have you already performed the same experiments on other cells suh as HepG2? They were very responsive to reactants used. But if I tell you about HL60...they had a very low basal flux (their normal respiration) and the responsiveness to oligomycin and FCCP was very restrained.
2. Is the ATP dependent respiratory activity of the tested cells significative?
3. No ATPase inhibitor substances or uncoupling agents are in the suspension media used or bacteria in cultures?
4. What about cells and mitochondria integrity during and after isolation and treatment of these two subjects?

[[email protected]]

Ib Therkelsen Arent

In our lab, a stock solution of 200 µM in 96% EtOH is kept in the freezer for months on end. To each Oxygraph chamber (2 ml) is added 5 µl during experiments, resulting in a final conc of 0,5 µM. With Isolated Mitos we never had a problem. With fibers I don't recall if we have ever used it. The Producers name I am not familiar with, but I might ask someone. [[email protected]]

ETS capacity after inhibition by Omy (2013-02-21)

• We are having problems recovering full activity with FCCP after oligomycin, and although diluting Omy to low but effective concentration help, it does not solve the problem. Thus, we are considering buying the more expensive forms of Omy. (2013-02-21; Doug Crawford, University of Miami, US)
• We are facing the same problems during recent years, and are worried about what is going wrong. Whereas pathologically stressed mitos with a latent injury do respond to the additional Omy induced stress by loosing ETS capacity, the controls gave us recently the same problems, which we consider as artefacts for unknown reasons. Quality control of formerly functional chemicals becomes an increasingly worrying issue. (Erich Gnaiger, Medical University Innsbruck, AT)

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