- SUIT protocol pattern: 1GM;2D;3P;4S;5U;6Rot-
The SUIT-014 protocols are designed for cells or tissue types that display a preference for GM over PM to support NADH-linked respiration. Adding pyruvate in the OXPHOS state enables to check if NADH-linked respiration can be further stimulated to determine N-pathway P capacity. SUIT-014 protocols provide an assessment of the linear coupling control (L- P- E) with NADH linked-substrates (PGM) and the control in ET state (N, NS, S), covering the contribution of two pathways which are most important in the mitochondria of many species, tissues and cell types.
Communicated by Cardoso LHD, Doerrier C, Huete-Ortega M, Gnaiger E (last update 2019-06-05)
Specific SUIT protocols
- SUIT-014 O2 pfi D042 for permeabilized fibers
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
- + NS-OXPHOS capacity provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity.
- + The presence of PGM and S establishes fully operative TCA cycle activity.
- + This protocol allows to analyse the convergence of pathways at the Q-junction (N, NS, S).
- + Mitochondrial external membrane integrity can be measured with the addition of cytochrome c. Application of the cytochrome c test early in the protocol ensures comparability of all states in case of any effect of c.
- - It was observed that addition of P (5 mM) after GM inhibited respiration in horse skeletal muscle fibres (Votion et al 2012), which was not the case when P was titrated in steps of 1 mM.
- - When evaluating the additive effect of the N- and S-pathway, it has to be considered that NSP- and NSE-capacities can only be compared with NP- and SE-capacities. This is not a problem when NSP = NSE (Gnaiger 2009). Otherwise, it may be assumed that SP = SE (Votion et al 2012), such that NSP can be compared with NP + SP. SUIT-004 should be chosen for the additive effect in the ET-state.
- - Rox may be lower in substrate states earlier in the SUIT protocol. Therefore, this Rox measurement is frequently taken as a methodological control rather than as the final basis of Rox correction of mitochondrial respiration (mt).
- - Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
- + This protocol can be extended with the Complex IV module, which can prolong the experimental time with ~30 min.
Compare SUIT protocols
|Lemieux 2017 Sci Rep||2017||Lemieux H, Blier PU, Gnaiger E (2017) Remodeling pathway control of mitochondrial respiratory capacity by temperature in mouse heart: electron flow through the Q-junction in permeabilized fibers. Sci Rep 7:2840, DOI:10.1038/s41598-017-02789-8.||Mouse||Heart|
|Schoepf 2016 FEBS J||2016||Schöpf B, Schäfer G, Weber A, Talasz H, Eder IE, Klocker H, Gnaiger E (2016) Oxidative phosphorylation and mitochondrial function differ between human prostate tissue and cultured cells. FEBS J 283:2181-96.||Human||Endothelial;epithelial;mesothelial cell|
Other cell lines
MitoPedia methods: Respirometry