SUIT-003 O2 ce D060: Difference between revisions
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|abbr= [[MitoKit-CII/Succinate-nv]] and [[MitoKit-CII/Malonate-nv]] | |abbr= [[MitoKit-CII/Succinate-nv]] and [[MitoKit-CII/Malonate-nv]] | ||
|description=[[File: | |description=[[File:ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama.png|400px]] | ||
|info='''B''' - [[coupling control protocol]] (CCP) with cell permeable compounds [[NV118/MitoKit-CII|NV118]] and [[NV161/MitoKit-CII|NV161]] -'''[[SUIT-003]]''' | |info='''B''' - [[coupling control protocol]] (CCP) with cell permeable compounds [[NV118/MitoKit-CII|NV118]] and [[NV161/MitoKit-CII|NV161]] -'''[[SUIT-003]]''' | ||
|application=O2 | |application=O2 | ||
|SUIT number=D060_ce1;ce2Omy;ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama | |SUIT number=D060_ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama | ||
}} | }} | ||
Revision as of 12:27, 26 June 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
SUIT-003 O2 ce-pce D060 is designed to study coupling control and plasma membrane permeability of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE, LEAK and ET. To study LEAK-respiration, the phosphorylation system is 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. The use of permeable compounds allows us to obtain further information about the mitochondrial respiratory capacities in living cells. The use of Snv after rotenone provides an idea about the S-pathway capacity in physiological conditions and then the addition of Mnanv and Antimycin A allows to obtain perform control over the succinate stimulation and to obtain ROX. The permeable compounds only work in living cells which plasma membrane has not been altered due that to be active required to be cleavage by esterases present in the cytosol.
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
Work in progress
- 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).
- CIV activity can also be measured after the cell viability test module.
- - 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