SUIT-003 O2 ce D060: Difference between revisions
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{{MitoPedia | {{MitoPedia | ||
|abbr= | |abbr= CCP-ce Snv,Mnanv | ||
|description=[[File:ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama.png|400px]] | |description=[[File:ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama.png|400px]] | ||
|info='''B''' - [[coupling control protocol]] | |info='''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). | ||
|application=O2 | |application=O2 | ||
|SUIT number=D060_ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama | |SUIT number=D060_ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama | ||
}} | }} | ||
: | {{Template:SUIT text D060}} | ||
__TOC__ | __TOC__ | ||
Communicated by [[Garcia-Souza LF]], [[Cardoso | Communicated by [[Garcia-Souza LF]], [[Cardoso LHD]] and [[Gnaiger E]] (last update 2019-09-24) | ||
== Representative traces == | |||
[[File:D060_O2_traces.png|600px]] | |||
{{Template:SUIT-003 O2 ce D060}} | {{Template:SUIT-003 O2 ce D060}} | ||
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== Strengths and limitations == | == Strengths and limitations == | ||
:::* It is recommended to run | :::* It is recommended to run control with carrier titrations in parallel, 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 | :::+ Addition of [[NV118/MitoKit-CII]] (Snv) and [[NV161/MitoKit-CII]] (Mnanv) allow studying the S-pathway in living cells. | ||
:::+ This is a | :::+ This is a [[coupling-control protocol]] in the living cells that provides information on [[ROUTINE]] and [[ET]]. | ||
:::- This protocol is provided without [[oligomycin]] addition. To also include [[LEAK]] in the protocol, an event for the addition of oligomycin and a related mark can be added by editing the DLP file, creating a [[Export DL-Protocol User (*.DLPU)|DLPU]]. However, the addition of [[oligomycin]] has to be carefully tested to avoid inhibitory effects in the evaluation of the [[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 [[Export DL-Protocol User (*.DLPU)|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. | :::- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler. | ||
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== Compare SUIT protocols == | == Compare SUIT protocols == | ||
::::* [[SUIT-003 O2 ce D061]] CCP with the addition of the [[Carrier control titrations|carrier]] DMSO instead of the plasma membrane-permeable compounds [[NV118/MitoKit-CII|Snv]] and [[NV161/MitoKit-CII|Mnanv]]. | ::::* [[SUIT-003 O2 ce D061]] CCP with the addition of the [[Carrier control titrations|carrier]] DMSO instead of the plasma membrane-permeable compounds [[NV118/MitoKit-CII|Snv]] and [[NV161/MitoKit-CII|Mnanv]]. | ||
::::* [[SUIT-003 O2 ce D050]] to study CI-deficiency with the plasma membrane-permeable succinate ([[MitoKit-CII/Succinate-nv]]) in living cells. | |||
::::* [[SUIT-003 O2 ce D028]] for cell membrane permeability test CCP(S). | ::::* [[SUIT-003 O2 ce D028]] for cell membrane permeability test CCP(S). | ||
::::* [[SUIT-003 O2 ce-pce D013]] first version of the | |||
::::* [[SUIT-003 O2 ce-pce D018]] second version of the | ::::* [[SUIT-003 O2 ce-pce D013]] first 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 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;ce2U-]] | ||
::::* [[SUIT-003 Ce1;ce2SD;ce3Omy;ce4U-]] | ::::* [[SUIT-003 Ce1;ce2SD;ce3Omy;ce4U-]] | ||
::::* [[SUIT-003 Ce1;ce2SD;ce3U;ce4Rot;ce5Ama]] | ::::* [[SUIT-003 Ce1;ce2SD;ce3U;ce4Rot;ce5Ama]] | ||
::::* [[SUIT-003 Ce1;ce2U;ce3Rot;ce4S;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). | ::::* [[SUIT-006]]: Coupling-control protocol for mt-preparations (mtprep), counterpart to SUIT-003 for non-permeabilized cells (ce). | ||
== Chemicals and syringes == | |||
{{Template:Chemicals SUIT-003 D060}} | |||
::: Suggested stock concentrations are shown in the specific DL-Protocol. | |||
Latest revision as of 14:34, 27 April 2021
Description
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
O2k-Application: O2
In SUIT-003 O2 ce D060 protocol, the use of permeable succinate and malonate enables further information to be obtained 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 enables evaluation of 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)
Representative traces
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
- It is recommended to run control with carrier titrations in parallel, 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 living cells.
- + This is a coupling-control protocol in the living cells 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 D050 to study CI-deficiency with the plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) in living cells.
- 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 |
ce4Rot | Rotenone (Rot) | |
ce5Snv | Succinate-nv (Snv) | |
ce6Mnasv | Malonate-sv (Mnasv) | |
ce7Ama | Antimycin A (Ama) |
- Suggested stock concentrations are shown in the specific DL-Protocol.
References
MitoPedia concepts: SUIT protocol, SUIT B, Find
MitoPedia methods:
Respirometry