SUIT-003 O2 ce D060: Difference between revisions
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::: '''[[MitoPedia: SUIT]]''' - 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). | ::: '''[[MitoPedia: SUIT]]''' - 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 protocol pattern]]:''' ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama | ::: '''[[SUIT protocol pattern]]:''' 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 [[NV118/MitoKit-CII| Snv]] after rotenone provides information about the [[S-pathway]] | 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 [[NV118/MitoKit-CII| 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 [[NV161/MitoKit-CII| Mnanv]] will counteract this effect. Moreover, the SUIT-003 O2 ce D060 protocol allows to evaluate the [[coupling control state |coupling control]] of living cells. Respiratory capacities are tested in a sequence of coupling states: [[ROUTINE]] and [[ET]]. 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-pathway|electron transfer system]] which would lead to an underestimation of [[ET-capacity]]. | ||
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Revision as of 16:56, 1 July 2019
Description
Abbreviation: MitoKit-CII/Succinate-nv and MitoKit-CII/Malonate-nv
Reference: B - coupling control protocol (CCP) with cell permeable compounds NV118 and NV161 -SUIT-003
SUIT number: D060_ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama
O2k-Application: O2
- MitoPedia: SUIT - 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 protocol pattern: 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 ET. 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 and Gnaiger E (last update 2019-06-26)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
ce1 | ROUTINE | ce1
| ||
(ce2Omy) | L(Omy) | ce1;(ce2Omy)
| ||
ce3U | E | ce1;(ce2Omy);ce3U
| ||
ce4Rot | ROX | ce1;(ce2Omy);ce3U;ce4Rot
| ||
ce5Snv | E | ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv
| ||
ce6Mnanv | ROX | ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv;ce6Mnanv
| ||
ce7Ama | ROX | ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama
|
Strengths and limitations
- The SUIT-003 O2 ce D060 is a cell coupling control protocol. The total cell count (Nce) is the sum of viable cells (Nvce) and dead cells (Ndce).
- + Addition of NV118/MitoKit-CII (Snv) and NV161/MitoKit-CII (Mnanv) allow to study the S-pathway in intact cells.
- - This protocol is provided without oligomycin addition. To also include LEAK in the protocol, an event for addition of oligomycin and a related mark can be added by edditing 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 cell permeable compound NV161 to test the succinate transport capacity through the plasma membrane.
- 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).
References
MitoPedia concepts: SUIT protocol, SUIT B, Find
MitoPedia methods:
Respirometry