From Bioblast
Description
Abbreviation: PM+S+Rot
Reference: A:
- SUIT-category: NS
- SUIT protocol pattern: orthogonal 1PM;2D;3S;4Rot;5U;-
This is a protocol to investigate oxygen flux N- and NS-pathway and S-pathway control state in the OXPHOS state and S-pathway control state in the ET state in isolated mitochondria and permeabilized cells. SUIT-031 Q mt D072 and SUIT-031 Q ce-pce D074 are designed to measure simultaneously O2 flux and the redox state of the Q-pool in isolated mitochondria and permeabilized cells.
Communicated by Komlodi T (names to be added) (last update 2021-02-08)
Specific SUIT protocols
SUIT-031 Q mt D072
- SUIT-031 Q mt D072 simultaneous determination of O2 flux and the redox ratio of the Q-pool in isolated mitochondria.
SUIT-031 Q ce-pce D074
- SUIT-031 Q ce-pce D074 simultaneous determination of O2 flux and the redox ratio of the Q-pool in permeabilized cells.
SUIT-031 O2 mt D075
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1PM | Pyruvate (P) and Malate (M) | |
2D | ADP (D) | |
3S | Succinate (S) | |
4Rot | Rotenone (Rot) | |
5U | Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) | Can be substituted for other uncoupler. |
6Ama | Antimycin A (Ama) |
SUIT-031 O2 ce-pce D000
Strengths and limitations
- + Reasonable duration of the experiment.
- NS-OXPHOS capacity provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity.
- A succinate concentration of >10 mM may be required for saturating SE capacity.
- + You obtain information in a single protocol about the NS-, S and N- pathway control state.
- - CIV activity cannot be determined and Cytochrome c test cannot be performed when measuring the Q redox state.
- - Careful washing is required after the experiment to avoid carry-over of the inhibitors and uncoupler.
- + Reasonable duration of the experiment.
Compare SUIT protocols
- SUIT-006 O2 mt D047 is a coupling-control protocol in the N-pathway for isolated mitochondria, tissue homogenate and cells permeabilized before addition to the O2k- chamber.
- SUIT-004 is designed to provide a quick assessment of the linear coupling control (L- P- E) with NADH-linked substrates (PM) and the contribution of the S-pathway to the ET state (N, NS, S).
- SUIT-008 is designed to assess the additivity between the N- and S-pathway in the Q-junction, providing a physiologically relevant estimate of maximum mitochondrial respiratory capacity.
References
MitoPedia concepts: MiP concept, SUIT protocol, Recommended
MitoPedia methods:
Respirometry