Reference: A: Coupling-control protocol, mtprep
- SUIT protocol pattern: 1X;2D;3Omy;4U;5Ama
The SUIT-006 protocol is a coupling-control protocol for mitochondrial preparations. Depending on the aims of the researcher, different combinations of substrates can be used to assess coupling control (L- P- E or L- P-L(Omy)- E) at a defined electron-transfer-pathway state. The use of oligomycin is optional, however, it provides important information when residual and endogenous adenylates are present (which may happen if ATPases are active in the sample). This situation may lead to overestimated LEAK respiration measured in the absence of adenylates - L(n). Therefore, oligomycin can be used to verify whether this occurs and obtain the LEAK state appropriately. Since higher concentrations of Omy can decrease the ET state induced upon addition of uncoupler, the required concentration of Omy has to be assessed by the Omy titration test.
In the DatLab software, SUIT-006 DLP files are currently provided for the categories N(PM) and S. For using this protocol with other substrate/inhibitor combinations, a personalized DLPU can be created. For O2 application with ce-pce, choose SUIT-006_O2_ce-pce_D029 to create the DLPU, for mt, choose SUIT-006 O2 mt D047. For O2 and H2O2 measurements (AmR), choose SUIT-006 AmR mt D048, and for O2 and membrane potential measurements, choose SUIT-006 Fluo mt D034. For O2 and Q redox state measurements, choose SUIT-006 Q mt D071 or SUIT-006 Q ce-pce D073.
Communicated by Cardoso LHD, Doerrier C, Komlodi T, Gnaiger E (last update 2021-11-25)
Specific SUIT protocols
SUIT-006 O2 ce-pce D029
- SUIT-006 O2 ce-pce D029 for N-pathway with permeabilized cells. Omy may be added in order to not overestimate LEAK respiration, that might be overestimated in 1PM if there are endogenous adenylates.
SUIT-006 O2 mt D047
- SUIT-006 O2 mt D047 for N-pathway with isolated mitochondria or tissue homogenate. Omy may be added in order to not overestimate LEAK respiration, that might be overestimated in 1PM if there are endogenous adenylates.
SUIT-006 AmR mt D048
- SUIT-006 AmR mt D048 to study simultaneously the O2 flux and H2O2 flux on isolated mitochondria, tissue homogenate and permeabilized cells, in particular to study the influence of mt-membrane potential on NADH-linked pathway initiated mt-H2O2 production.
SUIT-006 Fluo mt D034
- SUIT-006 Fluo mt D034 to study simultaneously the O2 flux and mitochondrial membrane potential on isolated mitochondria, tissue homogenate and permeabilized cells.
SUIT-006 MgG mt D055
- SUIT-006 MgG mt D055 to study simultaneously the O2 flux and mitochondrial ATP production on isolated mitochondria, tissue homogenate and cells permeabilized previously to addition to the O2k-chamber.
SUIT-006 O2 mt D022
- SUIT-006 O2 mt D022 for S-pathway with isolated mitochondria, tissue homogenate and or in cells permeabilized previously to addition to the O2k-chamber.
SUIT-006 Q mt D071
- SUIT-006 Q mt D071 to study simultaneously the O2 flux and the Q-redox state in isolated mitochondria or in cells permeabilized previously to addition to the O2k-chamber.
SUIT-006 Q ce-pce D073
- SUIT-006 Q ce-pce D073 to study simultaneously the O2 flux and the Q-redox state in permeabilized cells.
Strengths and limitations
- This protocol is indicated when the aim is to analyse coupling control in mtprep (see MitoPedia: Sample preparations) at a defined electron-transfer-pathway state.
- + Different combinations of substrates and inhibitors (X) can be used depending on the specific aims, e.g.: PGM, PM, GM, MnaPM or other combinations for N-pathway control state; S or RotS for S-pathway control state.
- + The combinations of substrates for one ET-pathway state with inhibitors for other pathways provide the best coupling-control protocols for one specific ET-pathway state, e.g.: PM in the presence of Mna for N-pathway control state, succinate in the presence of rotenone for S-pathway control state. In the specific case of S-pathway control state, rotenone should be added to avoid reverse electron transfer (RET).
- + Reasonable duration of the experiment.
- + This protocol can be extended with the Complex IV module.
- - 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, if applied (e.g., rotenone&succinate, SRot-pathway control state).
- - LEAK respiration in the absence of Omy may be overestimated due to ATP turnover stimulated by endogenous adenylates. This can be evaluated by SUIT 1X;2D;3Omy;4U;-. In mouse brain homogenate with succinate&rotenone, L(n) and L(Omy) are identical (Krumschnabel et al 2014).
- - CIV activity and Cytochrome c test cannot be performed together with the fluorescence (SUIT-006 AmR mt D048, SUIT-006 AmR mt D034 and SUIT-006 MgG mt D055).
Compare SUIT protocols
- SUIT-001: Extended protocol (Reference protocol 1) with SUIT-006 as the initial coupling control module (with PM as NADH-linked substrates).
- SUIT-004: Shortened version of SUIT-001, also with SUIT-006 as the initial coupling control module (with PM as NADH-linked substrates).
- SUIT-003: Coupling-control protocol for non-permeabilized cells (ce), counterpart to SUIT-006 for mt-preparations (mtprep).
|MiPNet26.10 MgG data analysis||2021-09-16|
|Cardoso 2021 BEC MgG||2021||Cardoso LHD, Doerrier C, Gnaiger E (2021) Magnesium Green for fluorometric measurement of ATP production does not interfere with mitochondrial respiration. https://doi.org/10.26124/bec:2021-0001||Mouse||Heart|
|Komlodi 2021 BEC AmR-O2||2021||Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. https://doi.org/10.26124/bec:2021-0004||Saccharomyces cerevisiae||Other cell lines|
|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. https://doi.org/10.1016/B978-0-12-416618-9.00009-1||Mouse||Nervous system|
MitoPedia concepts: MiP concept, SUIT protocol, Recommended
MitoPedia methods: Respirometry, Fluorometry