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

From Bioblast
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{{MitoPedia
{{MitoPedia
|abbr=
|abbr= [[MitoKit-CII/Succinate-nv]] and [[MitoKit-CII/Malonate-nv]]
|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]]''' ย 
|info='''B''' - [[coupling control protocol]] (CCP) with cell permeable compounds [[NV118/MitoKit-CII|NV118]] and [[NV161/MitoKit-CII|NV161]] -'''[[SUIT-003]]''' ย 
|application=O2
|SUIT number=D060_ce1;ce2Omy;ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama
}}
}}
::: '''[[SUIT protocol pattern]]:''' ce1;ce2Omy;ce3U;ce4Rot;ce5Snv;ce6Mnanv;ce7Ama




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ย  Communicated byย  [[Garcia-Souza LF]] and [[Gnaiger E]] (last update 2019-06-24)
ย  Communicated byย  [[Garcia-Souza LF]] and [[Gnaiger E]] (last update 2019-06-26)





Revision as of 10:07, 26 June 2019


high-resolution terminology - matching measurements at high-resolution


SUIT-003 O2 ce D060

Description

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

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)


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

Work in progress
- 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


References

MitoPedia concepts: SUIT protocol, SUIT B, Find 


MitoPedia methods: Respirometry