SUIT-003 O2 ce D060

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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 allows to obtain further information 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 allows to evaluate 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. NeuroVive #: NV118
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. NeuroVive #: NV161
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 in a parallel a control with carrier titrations, 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 intact cells.
+ This is a cell coupling-control protocol 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 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