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MiPNet discussion forum: Respiratory responses to Rotenone addition (2019-04-26)

Oroboros Instruments

The observation:

Using a coupling control protocol with intact cells, e.g. SUIT-003 ( or related SUIT protocols, the addition of Rotenone often inhibits respiration to the same extent as seen after subsequent Antimycin A addition. However, in some cell types and/or after some treatments the inhibition by Rotenone is clearly less pronounced than the inhibition after subsequent Antimycin A addition.

The problem:

It is not clear what this difference (inhibition by Rotenone vs. inhibition by Rotenone + Antimycin) signifies.

To obtain insight into this, we believe it could be helpful to collect examples from users with different cells, treatments, media (which substrates are contained in these) and whatever else condition might have an impact in this situation.

We thus ask you to share your experience and to send a description of the conditions (cells, medium, substrates, pre-treatment…) and ideally (a) DatLab file(s) showing the observation. We will then summarize these observations and make them available to everyone on our website for further discussion.

Rot-Ama intact cells.png

Example: HeLa cells examined with a simple Coupling Control Protocol. As in many cells, Rotenone inhibits respiration to a large extent and addition of Antimycin A has only little further inhibitory effect. Hela cells; 2 mL chamber; 0.455*10^6 cells/mL; 37°C; MiR05; no external substrates. [Exp.: 2018-12-04 PS4-02.DLD]

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Q & A

Q: I want to make a 1 mM stock solution in EtOH but it doesn't dissolve very good. Do you remember how you did it? Did you heat it up?
A: Yes, you should heat it up - best in a water bath, and the best would be to stirr it simultaneously with a magnetic stirrer.