Permeabilized muscle fibers
References
The following references describe in detail the basis for working with muscle fibers in the O2k.
- Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810: 25-58.
- MiPNet14.14 PermeabilizedFibrePreparation|Preparation of permeabilized muscle fibres for diagnosis of mitochondrial respiratory function. Mitochondr Physiol Network 14.14.]]
- Isolated mitochondria or permeabilized tissues and cells. Mitochondr. Physiol. Network 11.05.
- Mitochondrial respiration medium - MiR06. Mitochondr Physiol Network 14.13.
Chemicals and Media
What is the optimum ADP concentration to stimulate maximum muscle fibre respiration?
Especially in muscle fibers an ADP concentration up to 5 mM needs to be applied to be in the saturating range, an ADP titration might be required to evaluate, whether the applied concentration is saturating.
Pesta 2012 Methods Mol Biol:At a high apparent Km for ADP of 0.5 mM, flux at 2.5 and 5 mM ADP is ADP-limited by 13% and 7% (assuming L/P=0.2). 2.5 mM ADP is saturating in many cases, yet a further increase of ADP concentration provides a test for saturating [ADP]. This is particularly important for evaluation of OXPHOS versus ETS capacity (P versus E; Fig. 5).
High oxygen concentration
Why is it necessary to measure muscle fiber respiration above air saturation?
Respiratory flux of muscle fibre bundles is limited by oxygen diffusion even above 50% air saturation (O2 concentration of 100 ΞΌM). This problem is carefully controlled and avoided by application of a high oxygen regime.
See: Pesta 2012 Methods Mol Biol, Gnaiger 2003 Adv Exp Med Biol.
