SUIT-001 O2 ce-pce D003

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SUIT-001 O2 ce-pce D003



Abbreviation: RP1 ce-pce

Reference: A - SUIT-001 - SUIT reference protocol Reference protocol RP1 for living cells (ce, ROUTINE respiration) and cells that are permeabilized in the 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 2020-09-03)

Representative traces

D003 O2 traces.png

MitoPedia: SUIT

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 ROX ce1;1Dig
  • Optimum effective digitonin concentration for complete plasma membrane permeabilization.
Step State Pathway Q-junction Comment - Events (E) and Marks (M)
2c PMcP N CI 1PM;2D;2c
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 entry into Q-junction Comment
## Azd ROX


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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:
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.
+ 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 can not be 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.
- Lengthy 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.


MiPNet21.06 SUIT RP2018-06-25
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. doi: 10.1007/978-1-4939-7831-1_3Human
Saccharomyces cerevisiae
Skeletal muscle
Endothelial;epithelial;mesothelial cell
Blood cells

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry