Reference: A: SUIT-017
SUIT number: D049_1OctM;2D;2c;3G;4S;5U;6Rot;7Ama
SUIT-017 O2 pfi D049 gives information on F-pathway in LEAK state and OXPHOS state avoiding fatty acid oxidation (FAO) overestimation in the presence of anaplerotic pathways. In addition, the pathway control of FN and FNS in OXPHOS state and of FNS and S in ET state is evaluated. Permeabilized muscle fibers are sensitive to oxygen supply due to limited diffusion of oxygen to the fiber bundle core. To counteract this limitation, hyperoxic conditions (400-250 µM O2) must be employed. To set the optimal oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration.
Communicated by Cardoso LHD, Antunes D and Gnaiger E (last update 2019-07-31)
Steps and respiratory states
|Step||State||Pathway||Q-junction||Comment - Events (E) and Marks (M)|
|Step||Respiratory state||Pathway control||ET-Complex entry into Q-junction||Comment|
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- Pathway control
Strengths and limitations
- Comparison of GM- with PM-capacity yields important information on N-pathway respiratory control upstream of CI (Lemieux et al. 2017; Votion et al. 2012).
- + Glutamate is easier to prepare compared to pyruvate.
- + Reasonable duration of the experiment.
- + This protocol can be extended with the Complex IV module, which can prolong the experimental time with ~30 min.
Compare SUIT protocols
Chemicals and syringes
|Step||Chemical(s) and link(s)||Comments|
|1OctM||Octanoylcarnitine (Oct) and Malate (M)|
|2c||Cytochrome c (c)|
|5U||Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)||Can be substituted for other uncoupler|
|7Ama||Antimycin A (Ama)|
- Suggested stock concentrations are shown in the specific DL-Protocol.
|Pesta 2012 Methods Mol Biol||2012||Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle. Methods Mol Biol 810:25-58.||Human||Skeletal muscle|
Other cell lines
MitoPedia methods: Respirometry