MiPNet03.02 Chemicals-Media

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O2k-Protocols
Selected media and chemicals for respirometry with mitochondrial preparations.

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Oroboros (2016-08-30) Mitochondr Physiol Network

Abstract: Fontana-Ayoub M, Fasching M, Gnaiger E (2016) Selected media and chemicals for respirometry with mitochondrial preparations. Mitochondr Physiol Network 03.02(18):1-10.

Different media for tissue preparation and respiration are used in investigations of mitochondrial function. Initial decisions on the composition of media and chemicals are decisive for long-term studies and crucial for comparability of results. As a guideline, we summarize an update of our experience with media and chemicals for high-resolution respirometry with isolated mitochondria, permeabilized cells, muscle fibres and tissue homogenates. Whereas optimization is necessary for specific experimental protocols, standardization will improve the comparability of results obtained in different laboratories. Efforts towards standardization are important for the advancement of mitochondrial physiology.

» Product: Oroboros O2k, O2k-Catalogue


O2k-Network Lab: AT Innsbruck Oroboros


Labels: MiParea: Instruments;methods 





HRR: O2k-Protocol 

O2k-chemicals and media 

Supplementary information

» MiPNet09.12 O2k-Titrations; MitoPedia
» Cytochrome c control factor
» For calculations, see Excel file: O2k-Titrations.xls
» For pH adjustment of BIOPS solution, note the temperature dependence:
  • at 0 °C pH = 7.1
  • at 23 °C pH = 6.75

History

  • Malate was listed for a final concentration of 2 mM until 2013. During this year, test experiments with mitochondria from various tissues and species and with different mt-preparations (isolated mitochondria, permeabilized fibres, tissue homogenate) revealed an inhibitory effect of 2 mM malate on CII linked respiration (S,Rot). The inhibitory effect is less at 0.5 mM malate, and 0.5 mM malate may be saturating for CI-linked respiration. Evaluation is required for the optimum malate concentrations in SUIT protocols with sequential CI, CI&II and CII substrate states.
  • Up to 2006, rotenone was added at a high final concentration (0.5 µM), hence a 0.5 mM stock solution was prepared. Since 0.05 µM may be fully inhibiting, we suggested to use a stock solution at lower concentration (0.1 mM), to reduce the problem of rotenone retention. The 2006 edition listed a 0.2 mM stock solution, but the amount added to 10 ml ethanol was given for a 0.15 mM stock solution. Our recent rotenone titrations with permeabilized muscle fibers, however, confirmed that the originally proposed higher rotenone concentration is actually required for full inhibition.