SUIT-003 O2 ce D061: Difference between revisions
No edit summary |
Garcia Luiz (talk | contribs) No edit summary |
||
Line 6: | Line 6: | ||
|SUIT number=D061_ce1;(ce2Omy);ce3U;ce4Rot;ce5DMSO;ce6DMSO;ce7Ama | |SUIT number=D061_ce1;(ce2Omy);ce3U;ce4Rot;ce5DMSO;ce6DMSO;ce7Ama | ||
}} | }} | ||
::: '''[[MitoPedia: SUIT]]''' - Coupling control protocol (CCP) with the carrier of the plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) and plasma membrane-permeable malonate (MitoKit-CII/Malonate-nv] in living cells (ce): DMSO. | ::: '''[[MitoPedia: SUIT]]''' - Coupling control protocol (CCP) with the carrier of the plasma membrane-permeable succinate ([MitoKit-CII/Succinate-nv]) and plasma membrane-permeable malonate ([MitoKit-CII/Malonate-nv]) in living cells (ce): DMSO. | ||
::: '''[[SUIT protocol pattern]]:''' ce1;(ce2Omy);ce3U;ce4DMSO;ce5DMSO;ce6Rot;ce7Ama | ::: '''[[SUIT protocol pattern]]:''' ce1;(ce2Omy);ce3U;ce4DMSO;ce5DMSO;ce6Rot;ce7Ama | ||
SUIT-003 O2 ce D061 protocol is the control of the [[SUIT-003 O2 ce D060]] adding the [[Carrier control titrations|carrier]] DMSO in [[ROX]] instead of the plasma membrane-permeable succinate ([[MitoKit-CII/Succinate-nv]])and plasma membrane-permeable malonate ([[MitoKit-CII/Malonate-nv | SUIT-003 O2 ce D061 protocol is the control of the [[SUIT-003 O2 ce D060]] adding the [[Carrier control titrations|carrier]] DMSO in [[ROX]] instead of the plasma membrane-permeable succinate ([[MitoKit-CII/Succinate-nv]]) and plasma membrane-permeable malonate ([[MitoKit-CII/Malonate-nv]). Moreover, the SUIT-003 O2 ce D061 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]]. | ||
__TOC__ | __TOC__ | ||
Communicated by [[Garcia-Souza LF]] and [[Gnaiger E]](last update 2019-06- | Communicated by [[Garcia-Souza LF]] and [[Gnaiger E]](last update 2019-06-27) | ||
{{Template:SUIT-003 O2 ce D061}} | {{Template:SUIT-003 O2 ce D061}} | ||
== Strengths and limitations == | == Strengths and limitations == | ||
:::* [[CIV]] activity can also be measured after the cell viability test module. | :::* The [[SUIT-003 O2 ce D060]] is a cell [[coupling control protocol]]. The total cell count (''N''<sub>ce</sub>) is the sum of viable cells (''N''<sub>vce</sub>) and dead cells (''N''<sub>dce</sub>). | ||
:::- | :::* This protocol can be extended with the cell viability test module (See CCV [[SUIT-003 O2 ce-pce D020]]). | ||
:::* [[CIV]] activity can also be measured after the antimycin A titration or after the cell viability test module. | |||
:::+ Reasonable duration of the experiment. | |||
:::- To obtain [[LEAK]] respiration, the concentration 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. | |||
Revision as of 17:19, 27 June 2019
Description
Abbreviation: Carrier control of the plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) and plasma membrane-permeable malonate (MitoKit-CII/Malonate-nv) - SUIT-003 O2 ce D060
Reference: B
SUIT number: D061_ce1;(ce2Omy);ce3U;ce4Rot;ce5DMSO;ce6DMSO;ce7Ama
O2k-Application: O2
- MitoPedia: SUIT - Coupling control protocol (CCP) with the carrier of the plasma membrane-permeable succinate ([MitoKit-CII/Succinate-nv]) and plasma membrane-permeable malonate ([MitoKit-CII/Malonate-nv]) in living cells (ce): DMSO.
- SUIT protocol pattern: ce1;(ce2Omy);ce3U;ce4DMSO;ce5DMSO;ce6Rot;ce7Ama
SUIT-003 O2 ce D061 protocol is the control of the SUIT-003 O2 ce D060 adding the carrier DMSO in ROX instead of the plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) and plasma membrane-permeable malonate ([[MitoKit-CII/Malonate-nv]). Moreover, the SUIT-003 O2 ce D061 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 2019-06-27)
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 | cce1;(ce2Omy);ce3U
| ||
ce4Rot | ROX | ce1;(ce2Omy);ce3U;ce4Rot
| ||
ce5DMSO | ROX | ce1;(ce2Omy);ce3U;ce4Rot;ce5DMSO
| ||
ce6DMSO | ROX | ce1;(ce2Omy);ce3U;ce4Rot;ce5DMSO;ce6DMSO
| ||
ce7Ama | ROX | ce1;(ce2Omy);ce3U;ce4Rot;ce5DMSO;ce6DMSO;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).
- This protocol can be extended with the cell viability test module (See CCV SUIT-003 O2 ce-pce D020).
- CIV activity can also be measured after the antimycin A titration or after the cell viability test module.
- + Reasonable duration of the experiment.
- - To obtain LEAK respiration, the concentration 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 D060 CCP with plasma membrane-permeable compounds MitoKit-CII/Succinate-nv and MitoKit-CII/Malonate-nv.
- 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