SUIT-009 AmR ce-pce D019

high-resolution terminology - matching measurements at high-resolution

SUIT-009 AmR ce-pce D019

Description

Abbreviation: H₂O₂ RET ce-pce S_L

Reference: B: short protocol for H₂O₂ (AmR) in ce: non-permeabilized cells, pce: permeabilized cells -SUIT-009

SUIT number: D019_ce1;1Dig;1S;2D;3P;4Rot;5Ama

SUIT-009 is a short protocol for simultaneous determination of O₂ and H₂O₂ flux in permeabilized cells. Living cells are added first to the O2k chamber and then permeabilized as a part of the protocol. Succinate (S) supports reverse electron flow from CII to CI (RET) with high H₂O₂ production in the LEAK state at low O₂ flow, S_L. Respiration is stimulated by ADP (OXPHOS capacity, S_P), while H₂O₂ flux declines dramatically. Pyruvate (P) supports the NADH-pathway and thus the convergent NS-pathway with little further effect on H₂O₂ flux, NS_P). Malate is not required, since oxaloacetate is formed from succinate-fumarate-malate. Antimycin A (Ama) inhibits Complex III (CIII) at the Q_o level inhibiting respiration to the level of Rox, and generally increases the H₂O₂ flux. The sensitivity of the Amplex UltraRed® (AmR) changes over experimental time and upon addition of chemicals. To correct H₂O₂ flux for the sensitivity changes, several H₂O₂ calibration steps are done during the experiment. The experimental control for O₂ flux in the absence AmR is carried out with SUIT-009 O2 ce-pce D016. According to our results, the H₂O₂ production is linearly dependent on the O₂ concentration in MiR05-Kit, therefore, during the measurements the O₂ concentration has to be well-defined. In this protocol, we suggest not to go under ~100 µM O₂. SUIT-018 AmR mt D041 has been designed to study O₂ dependence of H₂O₂ flux in mitochondrial preparations.

Communicated by Timon-Gomez A, Huete-Ortega M, Cardoso LHD, Doerrier C, Komlodi T, Gnaiger E (last update 2024-09-19)

Representative traces

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
0DTPA				DTPA is an iron chelator, which decreases the chemical fluorescence background created by the Amplex UltraRed assay. Administration of DTPA into the O2k-chamber is recommended before all other chemicals because the iron chelation capacity of the compound is time-dependent (approx. 10-15 min). However, the experiments can be carried out in the absence of DTPA.
0SOD				SOD or superoxide dismutase converts the anion superoxide released by the mitochondria into H₂O₂, making it accessible to the Amplex UltraRed assay.
0HRP				HRP or horseradish peroxidase catalyses the conversion of Amplex UltraRed and H₂O₂ towards the fluorescent resorufin.
0AmR				AmR or Amplex UltraRed forms resorufin with H₂O₂ catalysed by horseradish peroxidase HRP.

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.
1Dig	REN			ce1;1Dig Optimum effective digitonin concentration for complete plasma membrane permeabilization.

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1S	S_L(n)	S	CII	1S Succinate, S ( type S-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	S_P	S	CII	1S;2D Succinate, S ( type S-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3P	NS_P	NS	CI&II	1S;2D;3P NADH-linked substrates (type N-pathway to Q). & Succinate, S ( type S-pathway to Q). Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4Rot	S_P	S	CII	1S;2D;3P;4Rot Succinate, S ( type S-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state. Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway.
5Ama	ROX			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

Information is obtained on respiration and ROS production in the S- and NS-pathway.
SUIT-009 O2 ce-pce D016 determination of O₂ flux on permeabilized cells as a control for SUIT-009 AmR ce-pce D019 to test the inhibitory effect of the fluorescence dye on the mt-respiration.
To study the inhibitory effect of the AmR assay on the respiration using living cells, the following protocols are recommended: SUIT-003 AmR ce D058 with AmR and SUIT-003 AmR ce D059 with carrier titration as a control. These two protocols should be done in parallel.

+ This protocol is designed to evaluate RET in the S-linked pathway in the LEAK state, which is decreased by ADP due to reduction of the protonmotive force, pmF.

+ Short duration of the experiment.

- CIV activity and cytochrome c test cannot be performed together with the fluorescence assay.

- The H₂O₂ flux will be much lower compared to that observed with isolated mitochondria, which might be explained by the high antioxidant capacity of cytosolic compounds.

- In the case of HEK293T cells, however, due to the high malate-linked anaplerotic capacity, malate is converted not only to oxaloacetate but also to pyruvate, such that addition of succinate alone supports NS-pathway capacity (no effect of pyruvate on OXPHOS capacity. Under these conditions, reverse electron transfer is inhibited in the LEAK state, which prevents excessively high H₂O₂ flow.

Compare SUIT protocols

SUIT-006 AmR mt D048 to investigate the dependence of H₂O₂ flux on the mt-membrane potential in the N-control state in isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber).
SUIT-018 is a short protocol to study the oxygen dependence of O₂ flux and H₂O₂ production in isolated mitochondria or tissue homogenate (except of liver).

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
H₂O₂	Hydrogen Peroxide (H₂O₂)

Step	Chemical(s) and link(s)	Comments
0DTPA	DTPA	This step can be skipped
0SOD	Superoxide Dismutase (SOD)
0HRP	Horseradish peroxidase (HRP)
0AmR	Amplex UltraRed (AmR)

Step	Chemical(s) and link(s)	Comments
1Dig	Digitonin (Dig)

Step	Chemical(s) and link(s)	Comments
1S	Succinate (S)
2D	ADP (D)
3P	Pyruvate (P)
4Rot	Rotenone (Rot)
5Ama	Antimycin A (Ama)

Suggested stock concentrations are shown in the specific DL-Protocol.

References

MitoPedia concepts: SUIT protocol, SUIT B, Find

MitoPedia methods: Fluorometry