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MiPNet21.06 SUIT RP

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MiPNet21.06 SUIT RP

Publications in the MiPMap
O2k-Protocols
SUIT reference protocol for OXPHOS analysis by high-resolution respirometry.

ยป [In progress]

Oroboros (2018-06-25) Mitochondr Physiol Network

Abstract: Doerrier C, Sumbalova Z, Gnaiger (2016) SUIT reference protocol for OXPHOS analysis by high-resolution respirometry. Mitochondr Physiol Network 21.06(01):1-12.

O2k-technical support and open innovation
Open the pdf document above.
ยป Current O2k-series: NextGen-O2k Series XB and O2k Series J
ยป Current software versions DatLab 8.0: MitoPedia: DatLab
ยป MitoPedia: SUIT reference protocol
ยป Instrument: Oroboros O2k, O2k-Catalogue


โ€ข O2k-Network Lab: AT_Innsbruck_Oroboros

SUIT-catg


SUIT reference protocol RP1


SUIT states: 1-3PM(LPcE) 4PGM 5PGMS 6OctPGMS 7S 8SGp 9ROX 10Tm 11ROX

MitoPedia: SUIT

Steps and respiratory states

SUIT-001

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
  • Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function.
  • Noncoupled electron transfer state, ET state, with ET capacity E.
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 1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp;9Ama
  • 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


Questions.jpg


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SUIT reference protocol RP2

SUIT states: 1ROX 2OctM(P) 2OctM(c) 3OctPM 4OctPGM 5OctPGMS 6OctPGMSGp 7E 8SGp 9ROX 10CIV 11ROX

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).


Questions.jpg


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Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

Harmonized SUIT protocols

RP1 and RP2 in mt-preparations

SUIT RP1 SUIT-RP1 SUIT RP2 SUIT-RP2

SUIT RP1:1PM;2D:2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd
SUIT RP2:1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd

RP1&RP2.png Harmonization between RP1 and RP2

RP1 and RP2 in cells

RP1 and RP2 can be assessed in cells after the selective plasma membrane permeabilization. Digitonin is a mild detergent that permeabilizes plasma membranes. The optimum effective digitonin concentration for complete plasma membrane permeabilization of cultured cells can be determined directly in a respirometric protocol (see Digitonin). The use of living cells allows the possibility to evaluate respiration in two different modules: intact cells and permeabilized cells. ROUTINE respiration (ce1) is obtained before digitonin is employed, whereas after the use of digitonin we can address different Electron-transfer-pathway states.

SUIT RP1 in cells: ce1;1Dig;1PM;2D:2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10Tm;11Azd
SUIT RP2 in cells: ce1;1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd

Reference protocol RP1&RP2 pce.png Harmonization between RP1 and RP2 in cells


RP1 and RP2 in PBMC and PLT

In PBMC and PLT the reference protocol RP1 is performed in the absense of Oct: SUIT 1 for PBMC and PTL

SUIT RP1 for PBMC and PLT: 1PM;2D;2c;3U;4G;5S;6Rot;7Gp;8Ama;9AsTm;10Azd
SUIT RP2: 1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd


Reference protocol RP1&RP2 pce PBMCs PTLs.png Harmonization between RP1 and RP2 in PBMC and PTL cells


Experimental details

Work in progress: pre-publication protocol

Saturating ADP concentrations

Different concentrations of ADP are sufficient to obtain maximum flux for estimating OXPHOS capacity. The typical range for isolated mitochondria is 1 to 2.5 mM, for permeabilized cells 1 to 5 mM, for permeabilized muscle fibres 2.5 to 10 mM.

SUIT-RP development

RP-T01

  • RP1: Compared to RP1-T01, move Oct titration after S.
  • RP2: Compared to RP2-T01, move U titration after Gp.

RP-T02

  • RP2: Compared to RP2-T02, move D titration after the sample. Titration of M.1 before Oct. M2 is added after Oct.

SUIT RP2 development

Cytochrome c test

  • In protocol RP2, the cytochrome c test might better be moved from a c-titration after pyruvate (P) to a titration after M2. Otherwise, there is a risk of underestimation of FAO in cases of a cytochrome c effect. On the other hand, at the low flux before titration of P, the sensitivity of the cytochrome c test may be low.
~ Gnaiger Erich 18:26, 23 January 2016 (CET)

Mark names

  • Even the highly abbreviated names of respiratory states have become too long for DatLab, where mark names are restricted to 8 digits. The visibility in DatLab of long mark names is restricted. The simplest solution for short mark names is the use of a numerical sequence, with the preceeding event name added for information.
~ Gnaiger Erich 16:50, 23 January 2016 (CET)


Pre-publication communication

This communication is a pre-publication, inviting critical feedback and comments. All feedback will be carefully documented and evaluated in terms of justification of co-authorship. We intend to finally publish this topic in a peer-reviewed journal as an original article, with reference to the pre-publication history including reviewer's reports.

Experiments in progress

The SUIT reference protocol is presently applied in permeabilized HEK cells, mouse heart isolated mitochondria, liver homogenate, permeabilized skeletal muscle (mouse and human), and human PBMC and PLT. - Gnaiger Erich 18:47, 19 January 2016 (CET)
K-Regio MitoFit

Further details

ยป Introduction: Gnaiger 2014 MitoPathways
ยป MitoPedia: SUIT
ยป Definition: Substrate-uncoupler-inhibitor titration
ยป Context: SUIT protocol library
ยป Abbreviations: MitoPedia


Labels: MiParea: Instruments;methods 



Preparation: Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria 

Regulation: Substrate  Coupling state: LEAK, OXPHOS, ET  Pathway: F, N, S, Gp, CIV, NS, Other combinations  HRR: Oxygraph-2k 

MitoPathways, MitoFitPublication, SUIT-001, SUIT-002, SUIT-001 O2 mt D001, SUIT-001 O2 pfi D002, SUIT-001 O2 ce-pce D003, SUIT-001 O2 ce-pce D004, SUIT-002 O2 mt D005, SUIT-002 O2 pfi D006, SUIT-002 O2 ce-pce D007, SUIT-002 O2 ce-pce D007a