SUIT-007

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SUIT-007

Description

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SUIT-category:

SUIT protocol pattern:

DLP applications

• SUIT-7 O2 ce-pce D13

References

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1G	GL	(N)	CI	1G NL if glutamate dehydrogenase is present in the sample studied (Glutamate-anaplerotic pathway control state). Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	GP	(N)	CI	1G;2D NP if glutamate dehydrogenase is present in the sample studied (Glutamate-anaplerotic pathway control state). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	GcP	(N)	CI	1G;2D;2c Addition of cytochrome c yields a test for integrity of the mtOM (cytochrome c control efficiency). Stimulation by added cytochrome c would indicate an injury of the mtOM and limitation of respiration in the preceding state without added c due to loss of cytochrome c. Typically, cytochrome c is added immediately after the earliest ADP-activation step (OXPHOS capacity P with saturating [ADP]). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3M	GMP	N	CI	1G;2D;2c;3M NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4U	GME	N	CI	1G;2D;2c;3M;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			1G;2D;2c;3M;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

• For determination of respirometric viability index, it is required cells with functional mitochondria from both intact and permeabilized (dead) cell population.

+ S-ET capacity can be assessed after the performance of the coupling control protocol (see SUIT-003). ce6S, 1Dig and 1c steps can be implemented without interfering other steps.

+ Respirometric viability test correlates with other viability methods.

+ CIV activity is measured.

- Cells with succinate transporter are not suitable for the viability test.

- Pathological profiles could hinder the viability test.

- Longer protocol in comparison to SUIT-003. Protocol average length of 2 hours.

- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.

Compare SUIT protocols

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 

MitoPedia methods: Respirometry