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Difference between revisions of "SUIT-003 O2 ce D060"

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{{MitoPedia
{{MitoPedia
|abbr=ce
|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]] (CCP) with cell permeable compounds [[NV118/MitoKit-CII|NV118]] and [[NV161/MitoKit-CII|NV161]] -'''[[SUIT-003]]''' Β 
|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).
|SUIT number=D060_ce1;ce2Omy;ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama
|application=O2
|application=O2
|SUIT number=D060_ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama
}}
}}
::: '''[[SUIT protocol pattern]]:''' ce1;ce2Omy;ce3U;ce*
{{Template:SUIT text D060}}


__TOC__


__TOC__
Communicated byΒ  [[Garcia-Souza LF]], [[Cardoso LHD]] and [[Gnaiger E]] (last update 2019-09-24)


Communicated byΒ  [[Garcia-Souza LF]] (last update 2019-05-21)
== 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 ==
Work in progress
:::* 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.
:::* 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>).
:::+ Addition of [[NV118/MitoKit-CII]] (Snv) and [[NV161/MitoKit-CII]] (Mnanv) allow studying the S-pathway in living cells.
:::* [[CIV]] activity can also be measured after the cell viability test module.
:::+ This is a [[coupling-control protocol]] in the living cells that provides information on [[ROUTINE]] and [[ET]].
:::- The addition of [[oligomycin]] has to be carefully tested to avoid inhibitory effects in the evaluation of the [[ET-capacity]].
:::- 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.




== Compare SUIT protocols ==
== Compare SUIT protocols ==
::::* [[SUIT-003 O2 ce D061]] CCP with cell permeable compound [[NV161/MitoKit-CII|NV161]] to test the succinate transport capacity through the plasma membrane.
::::* [[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.
Β 
::::* [[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 Coupling control protocol and respirometric cell viability test (CCV protocol).
Β 
::::* [[SUIT-003 O2 ce-pce D018]] second version of the Coupling control protocol and respirometric cell viability test (CCV protocol).
::::* [[SUIT-003 O2 ce-pce D013]] first version of the coupling-control protocol and respirometric cell viability test (CCV protocol).
::::* [[SUIT-003 Ce1;ce2P;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-]] Β 
::::* [[SUIT-003 Ce1;ce2SD;ce3Omy;ce4U-]] Β 
::::* [[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).
Β 
== Chemicals and syringes ==
{{Template:Chemicals SUIT-003 D060}}
::: Suggested stock concentrations are shown in the specific DL-Protocol.





Latest revision as of 13:34, 27 April 2021


high-resolution terminology - matching measurements at high-resolution


SUIT-003 O2 ce D060

Description

Ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama.png

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

D060 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

Ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
ce1 ROUTINE ce1
  • ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.


(ce2Omy) L(Omy) ce1;(ce2Omy)
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
  • The addition of oligomycin is optional depending on the experimental question to resolve.
ce3U E ce1;(ce2Omy);ce3U
ce4Rot ROX ce1;(ce2Omy);ce3U;ce4Rot
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration.


ce5Snv E ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv
  • Noncoupled electron transfer state, ET state, with ET capacity E.
  • MitoKit-CII/Succinate-nv (diacetoxymethyl succinate) is a plasma membrane-permeable prodrug (permeable succinate; Snv) that diffuses across the plasma membrane. Cleavage of diacetoxymethyl residues is mediated by intracellular esterases, thus releasing succinate in the intracellular space. Abliva #: 01-118-s4
ce6Mnanv ROX ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv;ce6Mnanv
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration.
  • MitoKit-CII/Malonate-nv (diacetoxymethyl malonate) is a plasma membrane-permeable prodrug (permeable malonate; Mnanv) that diffuses across the plasma membrane. Cleavage of diacetoxymethyl residues is mediated by intracellular esterases, thus releasing malonate in the intracellular space. Abliva #: 01-161-s2
ce7Ama ROX ce1;(ce2Omy);ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration.


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

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