Abbreviation: CCP mt S(Rot)
Reference: A - SUIT-006 - Protocol for coupling control assessment in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)
SUIT number: D022_1SRot;2D;2c;(3Omy);4U;5Ama
SUIT-006 O2 mt D022 is a coupling-control protocol for mitochondrial preparations (tissue homogenate, isolated mitochondria and permeabilized cells). Different coupling control states are assessed (L(n) - P - E) at succinate pathway control state. The use of rotenone, that inhibits complex I, provides a specific coupling-control protocol for S-pathway, avoiding reverse electron transfer (RET).
NOTE: In the DatLab software, the DLP file for SUIT-006 O2 mt D022 is provided without the addition of oligomycin. For using this protocol with oligomycin addition, a DLPU can be created, with the Omy event and mark. Using oligomycin in the preliminary assays with a not yet studied model is useful to determine whether in this kind of sample LEAK respiration in the absence of ADP (1SRot) may be overestimated due to the presence of residual and endogenous adenylates.
Communicated by Cardoso LH, Komlodi T, Huete-Ortega M, Doerrier C and Gnaiger E (last update 2019-06-06)
Steps and respiratory states
|Step||State||Pathway||Q-junction||Comment - Events (E) and Marks (M)|
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- Pathway control
Strengths and limitations
- + The combination of succinate with rotenone, that inhibits complex I, provides the best coupling-control protocol specific for S-pathway, avoiding reverse electron transfer (RET).
- + Reasonable duration of the experiment.
- + This protocol can be extended with the Complex IV module.
- - The DLP for SUIT-006 O2 mt D022 is provided with the oligomycin step skipped. However, this is a good control to analyse LEAK respiration. In samples that may contain endogenous adenylates and active ATPases, L(n) respiration may be overestimated. Therefore, in these conditions, L(Omy) is a better estimative of LEAK respiration. A DLPU can be created with the addition of oligomycin. It must be taken in consideration that high concentrations of oligomycin may affect the analysis of ET capacity, therefore the concentration applied must be previously tested.
- - This protocol does not include internal ET-pathway control steps.
- - Careful washing is required after the experiment to avoid carry-over of uncoupler and inhibitors.
Compare SUIT protocols
Chemicals and syringes
|Step||Chemical(s) and link(s)||Comments|
|1SRot||Succinate (S) and Rotenone (Rot)|
|2c||Cytochrome c (c)|
|(3Omy)||Oligomycin (Omy)||This step can be skipped|
|4U||Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)||Can be substituted for other uncoupler|
|5Ama||Antimycin A (Ama)|
- Suggested stock concentrations are shown in the specific DL-Protocol.
|Krumschnabel 2014 Methods Enzymol||2014||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.||Mouse||Nervous system|
MitoPedia methods: Respirometry