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SUIT-001 O2 ce-pce D003

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high-resolution terminology - matching measurements at high-resolution


SUIT-001 O2 ce-pce D003

Description

SUIT-RP1

Abbreviation: RP1 ce-pce

Reference: A - SUIT-001 - SUIT reference protocol Reference protocol RP1 for living cells (ce, ROUTINE respiration) and cells permeabilized in the respirometric chamber (pce)

SUIT number: D003_ce1;1Dig;1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd

O2k-Application: O2

The SUIT-001 O2 ce-pce D003 protocol in combination with SUIT-002 O2 ce-pce D007 is specially designed to provide 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-001 O2 ce-pce D003 gives information on ROUTINE respiration (ce: non-permeabilized cells) and after the plasma membrane permeabilization (pce: permeabilized cells) on linear coupling control (L- P- E) with NADH linked-substrates (PM). Moreover, the pathway control in ET state (N, NS, FNS, S and SGp pathways) can be evaluated by using this SUIT protocol. SUIT-001 O2 ce-pce D003 can be extended with the CIV assay module.

Communicated by Doerrier C and Gnaiger E (last update 2022-07-07)

Representative traces

D003 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

SUIT-001

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 PML(n) N CI 1PM
2D PMP N CI 1PM;2D
2c PMcP N CI 1PM;2D;2c
3U PME N CI 1PM;2D;3U
4G PGME N CI 1PM;2D;3U;4G
5S PGMSE NS CI&II 1PM;2D;3U;4G;5S
6Oct OctPGMSE FNS FAO&CI&II 1PM;2D;3U;4G;5S;6Oct
  • 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.
  • Noncoupled electron transfer state, ET state, with ET capacity E.
7Rot SE S CII 1PM;2D;3U;4G;5S;6Oct;7Rot
8Gp SGpE SGp CII&GpDH 1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp
  • 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.
9Ama 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).
Step Respiratory state Pathway control ET-Complex Comment
## AsTm AsTmE CIV CIV
## Azd CHB


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Strengths and limitations

  • SUIT-001 in combination with SUIT-002 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 to:
1. obtain reference values;
2. evaluate mitochondrial physiological diversity, generating a mt-database on comparative mitochondrial physiology;
3. screen specific defects.
  • SUIT-001 and SUIT-002 are used in the MitoFit Proficiency test for inter-individual and inter-laboratory reproducibility as quality control.
  • A succinate concentration of >10 mM may be required for saturating SE capacity.
+ Linear coupling control (L-P-E) with N substrates (PM).
+ Pathway control in ET state (N, NS, FNS, S and SGp pathways).
+ Harmonization with SUIT-002 allows to perform both SUIT protocols in parallel. The cross-linked respiratory states can be statistically used as repeated measurements.
+ In cells, SUIT-001 and SUIT-002 harmonize in: ROUTINE (ce1), SGpE (8Gp in SUIT-001 and 10Rot in SUIT-002) and Complex IV.
+ Harmonization with many SUIT protocols (up to step 5S).
+ Oct is added in ET state to avoid uncoupler effect with fatty acids.
+ If NS- and FNS-pathways are similar (NSE ~ FNSE), the additivity index of N- and S-pathways in ET state can be evaluated.
+ 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 is not obtained in SUIT-001 O2 mt D001 where the cells added to the chambers are already permeabilized.
- The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT-001 in favour of measuring SE.
- Long duration of the experiment.

Compare SUIT protocols

  • SUIT-001 O2 ce-pce D004: SUIT-001 O2 ce-pce D003 (general SUIT-001 for permeabilized cells) differs from SUIT-001 O2 ce-pce D004 (SUIT-001 for permeabilized PBMC and PLT) in the presence and absence of octanoylcarnitine, respectively.
  • SUIT-002 O2 ce-pce D007 (RP2): 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-001 O2 mt D001: SUIT-001 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).
  • SUIT-004: Short version of RP1; without G, F and Gp.
  • SUIT-006: Very short initial coupling control module.

Chemicals and syringes

Step Chemical(s) and link(s) Comments
1Dig Digitonin (Dig)
Step Chemical(s) and link(s) Comments
1PM Pyruvate (P) and Malate (M)
2D ADP (D)
2c Cytochrome c (c)
3U Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) Can be substituted for other uncoupler
4G Glutamate (G)
5S Succinate (S)
6Oct Octanoylcarnitine (Oct)
7Rot Rotenone (Rot)
8Gp Glycerophosphate (Gp)
9Ama 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
Baglivo 2022 MitoFit-QC2022Baglivo E, Cardoso LHD, Cecatto C, Gnaiger E (2022) Statistical analysis of instrumental reproducibility as internal quality control in high-resolution respirometry. https://doi.org/10.26124/mitofit:2022-0018.v22022-08-04 published in Bioenerg Commun 2022.8.HEK
Baglivo 2022 BEC2022Baglivo E, Cardoso LHD, Cecatto C, Gnaiger E (2022) Statistical analysis of instrumental reproducibility as internal quality control in high-resolution respirometry. Bioenerg Commun 2022.8. https://doi.org/10.26124/bec:2022-0008HEK
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