Abbreviation: CCP-ce Snv,Mnanv
Reference: B - coupling-control protocol for living cells with cell permeable compounds MitoKit-CII/Succinate-nv and MitoKit-CII/Malonate-nv -SUIT-003 - Coupling-control protocol (CCP) with plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) and plasma membrane-permeable malonate (MitoKit-CII/Malonate-nv) in living cells (ce).
SUIT number: D060_ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama
In SUIT-003 O2 ce D060 protocol, the use of permeable succinate and malonate allows to obtain further information about the mitochondrial respiratory capacities in living cells. The plasma membrane-permeable compounds NV118/MitoKit-CII (Snv) and NV161/MitoKit-CII (Mnanv) require cleavage by esterases present in the cytosol to release respectively succinate and malonate, which will happen therefore only in living cells with intact plasma membrane. The use of Snv after rotenone provides information about the S-pathway in physiological conditions. Rotenone inhibits Complex I and consequently the TCA cycle and respiration in living cells, but intracellular release of succinate from the permeable compound may lead to stimulation of respiration. Afterwards, the addition of Mnanv will counteract this effect. Moreover, the SUIT-003 O2 ce D060 protocol allows to evaluate the coupling control of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE and Electron transfer pathway. Optionally, to study LEAK respiration, the phosphorylation system might be inhibited by oligomycin. The final concentration of oligomycin has to be carefully optimized for various cell types, to minimize the inhibitory effect on the electron transfer system which would lead to an underestimation of ET capacity.
Communicated by Garcia-Souza LF, Cardoso LHD and Gnaiger E (last update 2019-09-24)
Steps and respiratory states
|Step||State||Pathway||Q-junction||Comment - Events (E) and Marks (M)|
Strengths and limitations
- It is recommended to run in a parallel a control with carrier titrations, since DMSO may affect living cells. SUIT-003 O2 ce D061 is provided with this purpose.
- + Addition of NV118/MitoKit-CII (Snv) and NV161/MitoKit-CII (Mnanv) allow studying the S-pathway in intact cells.
- + This is a cell coupling-control protocol that provides information on ROUTINE and ET.
- - This protocol is provided without oligomycin addition. To also include LEAK respiration in the protocol, an event for the addition of oligomycin and a related mark can be added by editing the DLP file, creating a DLPU. However, the addition of oligomycin has to be carefully tested to avoid inhibitory effects in the evaluation of the ET capacity.
- - Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
Compare SUIT protocols
- SUIT-003 O2 ce D061 CCP with the addition of the carrier DMSO instead of the plasma membrane-permeable compounds Snv and Mnanv.
- SUIT-003 O2 ce D028 for cell membrane permeability test CCP(S).
- SUIT-003 O2 ce-pce D013 first version of the Coupling-control protocol and respirometric cell viability test (CCV protocol).
- SUIT-003 O2 ce-pce D018 second version of the Coupling-control protocol and respirometric cell viability test (CCV protocol).
- SUIT-003 Ce1;ce1P;ce3U;ce4Glc;ce5M;ce6Rot;ce7S;1Dig;1c;2Ama;3AsTm;4Azd coupling-control protocol and respirometric cell viability test (CCV protocol) without oligomycin.
- SUIT-003 Ce1;ce2U-
- SUIT-003 Ce1;ce2SD;ce3Omy;ce4U-
- SUIT-003 Ce1;ce2SD;ce3U;ce4Rot;ce5Ama
- SUIT-003 Ce1;ce2U;ce3Rot;ce4S;ce5Ama
- SUIT-006: Coupling-control protocol for mt-preparations (mtprep), counterpart to SUIT-003 for non-permeabilized cells (ce).
Chemicals and syringes
|Step||Chemical(s) and link(s)||Comments|
|(ce2Omy)||Oligomycin (Omy)||This step can be skipped|
|ce3U||Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)||Can be substituted for other uncoupler|
|ce7Ama||Antimycin A (Ama)|
- Suggested stock concentrations are shown in the specific DL-Protocol.
MitoPedia methods: Respirometry