SUIT-021

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SUIT-021

Description

1GM;2D;3S;4Rot;5Omy;6U-.png

Abbreviation: OXPHOS (GM+S+Rot+Omy)

Reference: A: simultaneous determination of O2 flux and mt-membrane potential

SUIT protocol pattern: 1GM;2D;3S;4Rot;5Omy;6U;-

This is a protocol to investigate the N- and NS-pathway control state on isolated mitochondria and tissue homogenate. The protocol can be applied to cells or tissue types that display a preference for GM over PM to support NADH-linked respiration. Oligomycin (Omy) is used to induce a LEAK state of respiration via the inhibition of the ATP synthase. Since higher concentrations of Omy can decrease the ET-capacity induced upon addition of uncoupler, the required concentration of Omy has to be determined. This is an ideal protocol for simultaneous determination of O2 flux and mitochondrial membrane potential on isolated mitochondria and tissue homogenate. SUIT-021 O2 mt D035 is a protocol which can be performed without the fluorescence dye as well.

Communicated by Huete-Ortega M, Komlodi T, Gnaiger E (last update 2019-06-05)

Specific SUIT protocols

SUIT-021 Fluo mt D036

1GM;2D;3S;4Rot;5Omy;6U;7Ama.png SUIT-020 Fluo mt D036 O2 DL-File.png SUIT-020 Fluo mt D036 DL-File.png

  • SUIT-021 Fluo mt D036 simultaneous determination of O2 flux and mt-membrane potential using any fluorescence dye (Safranin, TMRM, etc) on isolated mitochondria and tissue homogenate.

SUIT-021 O2 mt D035

1GM;2D;2c;3S;4Rot;5Omy;6U;7Ama.png SUIT-020 O2 mt D035 DL-File.png

  • SUIT-021 O2 mt D035 determination of O2 flux on isolated mitochondria and tissue homogenate as a control for SUIT-021 Fluo mt D036.


MitoPedia: SUIT

Steps and respiratory states

1GM;2D;2c;3S;4Rot;5Omy;6U;7Ama.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1GM GML(n) N CI 1GM
2D GMP N CI 1GM;2D
2c GMcP N CI 1GM;2D;2c
  • NADH-linked substrates (type N-pathway to Q).
  • Addition of cytochrome c yields a test for integrity of the mtOM. Stimulation by added cytochrome c would indicate an injury of the mtOM and limitation of respiration in the preceding state without added c due to loss of cytochrome c. Typically, cytochrome c is added immediately after the earliest ADP-activation step (P, OXPHOS-capacity with saturating [ADP]).
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
3S GMSP NS CI&II 1GM;2D;2c;3S
  • Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function.
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
4Rot SP S CII 1GM;2D;2c;3S;4Rot
5Omy SL(Omy) 1GM;2D;2c;3S;4Rot;5Omy
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
6U* SE S CII 1GM;2D;2c;3S;4Rot;5Omy;6U
7Ama ROX 1GM;2D;2c;3S;4Rot;5Omy;6U;7Ama
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of Antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).
Step Respiratory state Pathway control ET-Complex entry into Q-junction Comment
## AsTm AsTmE CIV CIV
## Azd ROX


Questions.jpg


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Strengths and limitations

  • NS-OXPHOS capacity provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity.
  • Comparison of GM- with PM-capacity yields important information on N-pathway respiratory control upstream of CI.
  • A succinate concentration of >10 mM may be required for saturating SE capacity.
  • Nigericin as a H++/K++ antiporter can be used to dissipate transmembrane pH gradient, which results in increased mt-membrane potential in the LEAK state.
+ You obtain information in a single protocol about the NS-, S and N- pathway control state.
+ The protocol can be combined with fluorescence dyes to evaluate mitochondrial membrane potential.
- Many fluorescence dyes (such as Safranin, TMRM etc) can inhibit components of the ET system, most commonly affecting NADH-linked respiration. Therefore, a control run of the protocol should be done in the absence of the fluorescence dye. The following protocols can be used: SUIT-021 O2 mt D035.
- CIV activity cannot be determined and Cytochrome c test cannot be performed together with the fluorescence dyes.
- Oligomycin concentration has to be determined. Higher concentrations of Oligomycin may inhibit the ET state.
- Careful washing is required after the experiment to avoid carry-over of the inhibitors and uncoupler.

Compare SUIT protocols

  • SUIT-001;SUIT-001 O2 mt D001for isolated mitochondria and tissue homogenate: a complex protocol to get information not only about the NS(PGM) in the OXPHOS state, but also in ET state and about the FGp-pathways additionally.
  • SUIT-011:it provides information about the NS(GM) pathway in the OXPHOS and ET-states, but without the addtion of Omy.
  • SUIT-014: it is similar protocol to SUIT-021 O2 mt D035, but with the addition of P in the OXPHOS state before S. Omy is not added in the protocol.

References

 YearReferenceOrganismTissue;cell
MiPNet20.13 Safranin mt-membranepotential2019-06-24
O2k-Protocols
O2k-FluoRespirometry: HRR and simultaneous determination of mt-membrane potential with safranin or TMRM.
MouseNervous system
Krumschnabel 2014 Methods Enzymol2014
O2k-Protocols contents
Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81.
MouseNervous system



MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry, Fluorometry