SUIT-006 MgG ce-pce D085

high-resolution terminology - matching measurements at high-resolution

SUIT-006 MgG ce-pce D085

Description

Abbreviation: CCP MgG ce-pce

Reference: A: short coupling-control protocol for simultaneous determination of O₂ flux and mitochondrial ATP production in permeabilized cells -SUIT-006

SUIT number: D085_ce1;1Dig;1PM;2D;3Cat;4U;5Ama

O2k-Application: MgG

MitoPedia: SUIT

SUIT protocol pattern: 1PM;2D;3Cat;4U;5Ama

SUIT-006 MgG ce-pce D085 is a protocol to investigate the O₂ flux and mitochondrial ATP production using the fluorescent dye Magnesium Green. In this protocol, the NADH Electron transfer-pathway state can be analyzed in permeabilized cells (permeabilized wby digitonin titration into the chamber). Addition of the substrates pyruvate & malate (PM) to the mitochondrial preparation leads to LEAK state, and further addition of ADP induces the OXPHOS state, in which respiration is coupled to ATP production. After ADP addition, the signal from MgG gradually decreases, reflecting the ANT-mediated exchange of ADP for ATP, the later one having higher affinity for Mg²⁺. This exchange is stopped by addition of the ANT inhibitor carboxyatractyloside (Cat), which leads to LEAK state. With the addition of uncoupler, the Electron transfer pathway state can also be assessed in this protocol.

SUIT-006 MgG ce-pce D085 protocol in DatLab 8 will be provided for N(PM) pathway. For using this protocol with other substrate/inhibitor combinations (e.g. succinate and rotenone for Succinate pathway control state or PMS for NS-pathway control state), a personalized DLPU can be created.

Communicated by Cardoso LHD (last update 2023-08-18)

Representative traces

Steps and respiratory states

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)
1PM	PM_L(n)	N	CI	1PM NADH-linked substrates (type N-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	PM_P	N	CI	1PM;2D NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3Cat	PM_L(Cat)	N	CI	1PM;2D;3Cat NADH-linked substrates (type N-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Cat), after blocking the adenine nucleotide translocase (ANT) with carboxyatractyloside.
4U	PM_E	N	CI	1PM;2D;3Cat;4U NADH-linked substrates (type N-pathway to Q). Uncoupler titration (avoiding inhibition by high uncoupler concentrations) to obtain electron transfer (ET) capacity E (noncoupled ET-state). Test for limitation of OXPHOS capacity P by the phosphorylation system (ANT, ATP synthase, phosphate transporter) relative to ET capacity E in mt-preparations: E-P control efficiency and E-L coupling efficiency. In living cells: E-R control efficiency and E-L coupling efficiency.
5Ama	ROX			1PM;2D;3Cat;4U;5Ama 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

Before performing this protocol, a calibration with the fluorescent dye and the determination of the K_d of ADP and ATP to Mg²⁺ in the pertaining experimental conditions need to be done. For more information, see: Magnesium Green.
The buffer for this assay must be chosen carefully. It can be performed in MiR05 prepared without MgCl₂ (with addition of 1 mM MgCl2 during the calibration or prior to the experiment) or with the ANT buffer (Chinopoulos 2014 Methods Enzymol).
Inhibitors of kinases and ATPases might be necessary depending on the mitochondrial preparation used, especially for permeabilized cells or tissue homogenates (Chinopoulos 2014 Methods Enzymol).
For protocols with MgG, ADP must be prepared without MgCl₂.

+ In this protocol, ATP production in OXPHOS can be assessed in parallel with respiration. The protocol also allows to analyze the coupling control (LEAK, OXPHOS and ET respiration) in the same ET-pathway control state.

- This protocol does not include internal ET-pathway control steps.

- Even though MgG seems not to inhibit respiration in isolated mitochondria, it is recommended to do an experimental control in the absence of the fluorescent dye to check for inhibitory effects. The following protocol can be used: SUIT-006 O2 mt D047.

- The test for mitochondrial outer membrane integrity with cytochrome c cannot be performed together with MgG assays.

The ATP synthase inhibitor oligomycin (Omy) can also be used, instead of carboxyatractyloside, to stop mitochondrial ATP production and induce LEAK state. However, high concentrations of Omy can decrease the ET capacity measured after addition of uncoupler, therefore the concentration must be tested.

Compare SUIT protocols

SUIT-006 MgG mt D055 for mitochondrial preparations such as isolated mitochondria, tissue homogenate, or cells permeabilized prior to addition to the chamber.

SUIT-006 O2 mt D047 for coupling control with pyruvate and malate as substrates.

SUIT-006 Fluo mt D034 coupling control with pyruvate and malate as substrates for analysing membrane potential and O₂ flux.

SUIT-006 AmR mt D048 coupling control with pyruvate and malate as substrates for analysing H₂O₂ flux and O₂ flux.

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
MgG	Magnesium Green
Mg	MgCl₂

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

Step	Chemical(s) and link(s)	Comments
1PM	Pyruvate (P) and Malate (M)	Can be substituted by other combination of substrates
2D	ADP (D) preparation for MgG assay	For this protocol, the ADP must be prepared without Mg²⁺ salts.
3Cat	Carboxyatractyloside (Cat)	Can be substituted for oligomycin
4U	SF6847	Can be substituted for other uncoupler
5Ama	Antimycin A (Ama)	Can be substituted for other CIII or CIV inhibitor

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

References

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Fluorometry