SUIT-003 AmR ce D058

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SUIT-003 AmR ce D058

Description

Abbreviation: ce

Reference: B: Coupling control in intact cells with oligomycin to study the inhibitory effect of the AmR assay components on the respiration ; ce: non-permeabilized cells -SUIT-003

SUIT number: D058_ce1;ce2Omy;ce3U;ce4Rot;ce5Ama

O2k-Application: AmR

MitoPedia: SUIT

SUIT-category: ce

SUIT protocol pattern: linear cell (ce) coupling control protocol ce1;ce2Omy;ce3U

SUIT-003 O2 ce D058 has been designed to study the inhibitory effect of the components of the Amplex UltraRed assay on coupling control state of intact cells. The different capacities are tested in the sequence OXPHOS, LEAK and ET. Also, in order to study the LEAK-respiration, we need to inhibit the phosphorylation system which in our protocol is achieved using oligomycin. However, the final concentration of oligomycin has to be carefully adjusted in preliminary experiments for different samples and, the inhibitory effect on the electron transfer system, has to be studied to avoid an underestimation of the ET-capacity. Superoxide dismutase and Horseradish peroxidase do not inhibit respiration, however, Amplex UltraRed seems to block at higher concentration ROUTINE respiratoin and the ET-capacity. The inhibitory effect of Amplex UltraRed should be tested with each sample type.

Communicated by  Komlodi T and Gnaiger E (last update 2019-05-15)

Steps and respiratory states

Step	State	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.
ce1SOD	ROUTINE	ce1;ce1SOD SOD or superoxide dismutase converts the anion superoxide released by the mitochondria into H₂O₂, making it accessible to the Amplex UltraRed assay.
ce1HRP	ROUTINE	ce1;ce1SOD;ce1HRP HRP or horseradish peroxidase catalyses the conversion of Amplex UltraRed and H₂O₂ towards the fluorescent resorufin.
ce1AmR	ROUTINE	ce1;ce1SOD;ce1HRP;ce1AmR AmR or Amplex UltraRed forms resorufin with H₂O₂ catalysed by horseradish peroxidase HRP.
ce2Omy	L_Omy	ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
ce3U	E	ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U Noncoupled electron transfer state, ET state, with ET capacity E.
ce4Rot	ROX	ce1;ce1SOD;ce1HRP;ce1AmR;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.
ce5Ama	ROX	ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U;ce4Rot;ce5Ama Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).

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Coupling control

» Coupling control state

» ET capacity

» OXPHOS capacity

» LEAK respiration

Pathway control

» Electron transfer pathway

» Fatty acid oxidation pathway control state, F

» NADH electron transfer-pathway state, N

» Succinate pathway control state, S

» NS-pathway control state, NS

» Glycerophosphate pathway control state, Gp

» Complex IV single step, CIV

» Anaplerotic pathway control state

Main fuel substrates

» Glutamate, G

» Glycerophosphate, Gp

» Malate, M

» Octanoylcarnitine, Oct

» Pyruvate, P

» Succinate, S

Glossary

» List of SUIT states

» SUIT concept

Strengths and limitations

This protocol has been designed to study the inhibitory effect of the Amplex UltraRed assay.
It is recommended to run this test with every differetn sample type.

+ Reasonable duration of the experiment.

- 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 D009 for non-permeabilized cells with oligomycin without the AmR assay
Protocols using AmR assay:

SUIT-006 AmR mt D048 to study simultaneously the O₂ flux and H₂O₂ flux on isolated mitochondria, tissue homogenate and permeabilized cells, in particular to study the influence of mt-membrane potential on NADH-linked pathway initiated mt-H2O2 production.
SUIT-009 AmR ce-pce D019 simultaneous determination of O₂ and H₂O₂ flux for non-permeabilized (ce) and permeabilized cells (pce; permeabilization of the plasma membrane occurs in the chambers through addition of digitonin )
SUIT-009 AmR mt D021 simultaneous determination of O₂ and H₂O₂ flux on isolated mitochondria and tissue homogenate
SUIT-013 AmR ce D023 for intact cells
SUIT-018 AmR mt D031 simultaneous determination of O₂ and H₂O₂ flux on isolated mitochondria, tissue homogenate and permeabilized cells at low oxygen concentration (tissue normoxia).
SUIT-018 AmR mt D040 simultaneous determination of O₂ and H₂O₂ flux on isolated mitochondria, tissue homogenate and permeabilized cells at high oxygen concentration (tissue hyperoxia).
SUIT-018 AmR mt D041 simultaneous determination of O₂ and H₂O₂ flux on isolated mitochondria, tissue homogenate and permeabilized cells changing the oxygen concentration in the same protocol.

References

MitoPedia concepts: SUIT protocol, SUIT B, Find

MitoPedia methods: Respirometry