SUIT-001 O2 ce-pce D004

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SUIT-001 O2 ce-pce D004

Description

SUIT-RP1 for PBMC and PLT

Abbreviation: RP1 ce-pce blood

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

SUIT number: D004_ce1;1Dig;1PM;2D;2c;3U;4G;5S;(6Oct);7Rot;8Gp;9Ama;10AsTm;11Azd

O2k-Application: O2


The SUIT-001 O2 ce-pce D004 protocol is the reference protocol 1 (RP1) specifically for PBMCs and PLTs. The main difference between the RP1 for PBMCs and PLTs and RP1 for other mitochondrial preparations ( see SUIT-001) is the absence of Oct in the case of blood cells. The SUIT-001 O2 ce-pce D004 protocol in combination with SUIT-002 O2 ce-pce D007a is specially designed to provide a common reference for comparison of respiratory control of PBMC and platelets cells (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol) in a wide variety of species. SUIT-001 O2 ce-pce D004 gives information of ROUTINE respiration (ce: non-permeabilized cells) and after the plasma membrane permeabilization (pce: permeabilized cells) of the linear coupling control (L- P- E) with NADH linked-substrates (PM). Moreover, the pathway control in ET state (N, NS, S and SGp pathways) can be evaluated by using this SUIT protocol. SUIT-001 O2 ce-pce D004 can be extended with the CIV assay module.

Communicated by Doerrier C and Gnaiger E (last update 2020-09-03)

Representative traces

D004 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

SUIT-001 for PBMCs and PLTs

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)
1PM PML(n) N CI PML or PM_L: Pyruvate & Malate, N-LEAK respiration, NL
2D PMP N CI 1PM;2D
2c PMcP N CI 1PM;2D;2c
  • NADH-linked substrates (type N-pathway to Q).
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
  • Addition of cytochrome c yields a test for integrity of the mtOM. 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 (P, OXPHOS capacity with saturating [ADP]).
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) 1PM;2D;3U;4G;5S;(6Oct)
  • Oct is skipped in PBMCs and PTLs
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
## AsTm AsTmE CIV CIV
## Azd ROX


Questions.jpg


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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 blood 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).
+ 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.
- Oct is not added in ET state.
- Lengthy duration of the experiment.

Compare SUIT protocols

  • SUIT-001 O2 ce-pce D004 (SUIT-001 for permeabilized PBMC and PLT) differs from SUIT-001 O2 ce-pce D003 (general SUIT-001 for permeabilized cells) in the absence and presence of octanoylcarnitine, respectively.
  • SUIT-002 O2 ce-pce D007a (RP2): Harmonized SUIT protocol for PBMC and PLT 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) Oct is skipped in PBMCs and PTLs
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
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.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