SUIT-002 O2 ce-pce D007

high-resolution terminology - matching measurements at high-resolution

SUIT-002 O2 ce-pce D007

Description

Abbreviation: RP2 ce-pce

Reference: A - SUIT-002 - SUIT reference protocol Reference protocol RP2 for living cells (ce, ROUTINE respiration) and cells that are permeabilized in the chamber (pce)

SUIT number: D007_ce1;1Dig;1D;2M;3Oct;3c;4M;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd

O2k-Application: O2

The SUIT-002 O2 ce-pce D007 protocol in combination with SUIT-001 O2 ce-pce D003 provides a common reference for comparison of respiratory control of permeabilized cells (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol) in a wide variety of cell types. SUIT-002 O2 ce-pce D007 is specially designed to give information on F-pathway in OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways. Moreover, the pathway control in OXPHOS state (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be evaluated by using this SUIT protocol. SUIT-002 O2 ce-pce D007 can be extended with the CIV assay module.

Communicated by Doerrier C and Gnaiger E (last update 2024-04-09)

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)
1D	REN			1D ADP is added to stimulate the consumption of endogenous fuel-substrates.
2M.1				Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
3Oct	OctM_P	F	FAO	1D;2M.1;3Oct Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration.
3c	OctMc_P	F	FAO	1D;2M.1;3Oct;3c Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state. 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]).
4M2	OctM_P	F(N)	FAO	1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. High concentration of malate, typically 2 mM, saturates the N-pathway. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
5P	OctPM_P	FN	F&CI	1D;2M.1;3Oct;4M2;5P NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
6G	OctPGM_P	FN	F&CI	1D;2M.1;3Oct;4M2;5P;6G NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
7S	OctPGMS_P	FNS	F&CI&II	1D;2M.1;3Oct;4M2;5P;6G;7S Respiratory stimulation by simultaneous action of the F-pathway, N-pathway, and S-pathway, with convergent electron flow in the FNS-pathway for reconstitution of TCA cycle function and additive or inhibitory effect of F. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
8Gp	OctPGMSGp_P	FNSGp	F&CI&II&GpDH	1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp Respiratory stimulation by simultaneous action of fatty acid (F), type N substrates (N), succinate (S), and glycerophosphate with convergent electron flow in the FNSGp-pathway to the Q-junction. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
9U	OctPGMSGp_E	FNSGp	F&CI&II&GpDH	1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U Respiratory stimulation by simultaneous action of fatty acid (F), type N substrates (N), succinate (S), and glycerophosphate with convergent electron flow in the FNSGp-pathway to the Q-junction. 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.
10Rot	SGp_E	SGp	CII&GpDH	1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot Respiratory stimulation by action of succinate and glycerophosphate, Gp, with convergent electron flow in the SGp-pathway (CII&GpDH-linked pathway to the Q-junction). Noncoupled electron transfer state, ET state, with ET capacity E.
11Ama	ROX			1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama 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).

Step	Respiratory state	Pathway control	ET-Complex	Comment
## AsTm	AsTm_E	CIV	CIV
## Azd	CHB

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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

SUIT-002 in combination with SUIT-001 provides a common reference for comparison of respiratory control in a large variety of species, tissues and cell types. Both SUIT protocols provide a mitochondrial mapping which allows:

1. to obtain reference values.

2. to evaluate mitochondrial physiological diversity, generating a mt-database on comparative mitochondrial physiology.

3. to screen specific defects.

SUIT-001 and SUIT-002 are used in the MitoFit Proficiency test for inter-individual and inter-laboratory reproducibility quality control.
A succinate concentration of >10 mM may be required for saturating S_E capacity.

+ SUIT-002 allows the depletion of endogenous substrates with ADP (1D).

+ The protocol provides information on F-pathway in OXPHOS state. The low concentration of malate used in this protocol, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.

+ F-pathway (3Oct-2M.1) can be compared to FN-pathway (5P) in OXPHOS state.

+ Pathway control in OXPHOS (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be observed.

+ Harmonization with SUIT-001 allows to perform both SUIT protocols in parallel. The cross-linked respiratory states can be statistically used as repeated measurements.

+ Harmonization with many SUIT protocols (up to step 7S).

+ In SUIT-002, the full set of pathways converging into Q (FNSGp) is obtained in OXPHOS and ET states. Therefore, P/E (8Gp/9U) at high ET capacity can be calculated.

+ This protocol can be extended with the Complex IV module.

+ The addition of non-permeabilized cells to the chambers provides additional information (ROUTINE respiration) which can not be obtained in SUIT-002 O2 mt D005 where the cells added to the chambers are already permeabilized.

- S-pathway in ET state is not obtained (it is obtained in SUIT-001).

- Lengthy duration of the experiment.

Compare SUIT protocols

SUIT-002 O2 ce-pce D007 (general SUIT-002 for permeabilized cells) differs from SUIT-002 O2 ce-pce D007a (SUIT-002 for permeabilized PBMC and PLT) in the concentration and volume of the titrations.

SUIT-001 O2 ce-pce D003 (RP1): Harmonized SUIT protocol for permeabilized cells (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol).

SUIT-002 O2 mt D005: SUIT-002 for permeabilized cells (cells added to the chamber have been permeabilized before their addition). This SUIT protocol provides no information on ROUTINE respiration of non-permeabilized cells (ce).

Chemicals and syringes

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

Step	Chemical(s) and link(s)	Comments
1D	ADP (D)
2M.1	Malate (M)
3Oct	Octanoylcarnitine (Oct)
3c	Cytochrome c (c)
4M2	Malate (M)
5P	Pyruvate (P)
6G	Glutamate (G)
7S	Succinate (S)
8Gp	Glycerophosphate (Gp)
9U	Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)	Can be substituted for other uncoupler
10Rot	Rotenone (Rot)
11Ama	Antimycin A (Ama)

Step	Chemical(s) and link(s)	Comments
## AsTm	Ascorbate (As) and TMPD (Tm)
## Azd	Azide (Azd)

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

References

	Year	Reference	Organism	Tissue;cell
MiPNet21.06 SUIT RP	2018-06-25	SUIT reference protocol for OXPHOS analysis by high-resolution respirometry.
Doerrier 2018 Methods Mol Biol	2018	Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. https://doi.org/10.1007/978-1-4939-7831-1_3	Human Mouse Rat Saccharomyces cerevisiae	Heart Skeletal muscle Endothelial;epithelial;mesothelial cell Blood cells HEK Platelet

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Respirometry