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SUIT-002 O2 mt D005

From Bioblast


high-resolution terminology - matching measurements at high-resolution


SUIT-002 O2 mt D005

Description

1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.png

Abbreviation: RP2 mt

Reference: A - SUIT-002 - SUIT reference protocol Reference protocol RP2 for isolated mitochondria, tissue homogenate and permeabilized cells

SUIT number: D005_mt;1D;2M;3Oct;3c;4M;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd

O2k-Application: O2


The SUIT-002 O2 mt D005 protocol in combination with SUIT-001_O2_mt_D001 provides a common reference for comparison of respiratory control of mitochondrial preparations such as isolated mitochondria, tissue homogenates and permeabilized cells (already permeabilized when they are added to the chamber) in a wide variety of species, tissues and cell types. SUIT-002 O2 mt D005 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 mt D005 can be extended with the CIV assay module.

Communicated by Doerrier C and Gnaiger E (last update 2019-09-24)

Representative traces

D005 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot-.png

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 1D;2M.1
3Oct OctMP 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 OctMcP 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 OctMP F(N) FAO 1D;2M.1;3Oct;3c;4M2
5P OctPMP FN FAO&CI 1D;2M.1;3Oct;4M2;5P
  • Respiratory stimulation by simultaneous action of the F-pathway and N-pathway with convergent electron flow in the FN-pathway for evaluation of an additive or inhibitory effect of F.
  • OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
6G OctPGMP FN FAO&CI 1D;2M.1;3Oct;4M2;5P;6G
  • Respiratory stimulation by simultaneous action of the F-pathway and N-pathway with convergent electron flow in the FN-pathway for evaluation of an additive or inhibitory effect of F.
  • OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
7S OctPGMSP FNS FAO&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 OctPGMSGpP FNSGp FAO&CI&II&GpDH 1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp
9U OctPGMSGpE FNSGp FAO&CI&II&GpDH 1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U
10Rot SGpE 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).


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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 SE 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.
- S-pathway in ET state is not obtained (it is obtained in SUIT-001).
- Lengthy duration of the experiment.
- We do not generally recommended the addition of already permeabilized cells, but we recommend to perform the permeabilization of the cell plasma membrane in the chambers (see SUIT-002 O2 ce-pce D007).

Compare SUIT protocols

  • SUIT-001 O2 mt D001 (RP1): Harmonized SUIT protocol for isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized).
  • SUIT-002 O2 ce-pce D007: SUIT-002 for non-permeabilized cells. Permeabilization of the cell plasma membrane occurs in the chambers through digitonin addition. Therefore, SUIT-002 O2 ce-pce D007 protocol provides information of non-permeabilized cell respiration (ce) and permeabilized cell respiration (pce).

Chemicals and syringes

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

 YearReferenceOrganismTissue;cell
MiPNet21.06 SUIT RP2018-06-25
O2k-Protocols
SUIT reference protocol for OXPHOS analysis by high-resolution respirometry.
Doerrier 2018 Methods Mol Biol2018Doerrier 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_3Human
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