MiPNet21.06 SUIT RP

SUIT reference protocol for OXPHOS analysis by high-resolution respirometry.

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OROBOROS (2016-01-23) Mitochondr Physiol Network

Abstract: Doerrier C, Sumbalova Z, Lamberti G, Krumschnabel G, Hiller E, Gnaiger (2016) SUIT reference protocol for OXPHOS analysis by high-resolution respirometry. Mitochondr Physiol Network 21.06(05):1-12. » Versions

» MitoPedia: SUIT reference protocol

» Instrument: O2k, O2k-Catalogue

• O2k-Network Lab: AT_Innsbruck_OROBOROS

Labels: MiParea: Instruments;methods

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

Regulation: Substrate Coupling state: LEAK, OXPHOS, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property.

HRR: Oxygraph-2k, O2k-Protocol

MitoPathways, MitoFitPublication

Work in progress

SUIT reference protocol-RP1

SUIT states: 1-4PM(LPcE) 5PGM 6PGMS 7PGMSOct 8S 9SGp 10ROX 11Tm 12ROX

Step	Respiratory state	Pathway control	Entry through electron transfer Complex
1PM	PM(L)	N	CI
2D	PM(P)	N	CI
3c	PM(P)c	N	CI
3NADH	PM(P)cNADH	N	CI
4U	PM(E)	N	CI
5G	PGM(E)	N	CI
6S	PGMS(E)	NS	CI&II
7Oct	PGMSOct(E)	NFS	CI&II&FAO
8Rot	S(E)	S	CII
9Gp	SGp(E)	SGp	CII&GpDH
10Ama		ROX	ROX
11Tm	Tm(E)	Tm	CIV
12Azd		ROX	ROX

SUIT reference protocol-RP2

SUIT states: 1ROX 2Oct(P) 3MOct(4c) 5PMOct 6PGMOct 7PGMSOct 8PGMSOctGp(9E) 10SGp 11ROX 12CIV 13ROX

Step	Respiratory state	Pathway control	Entry through electron transfer Complex
1D		ROX	ROX
2Oct	Oct(P)	(F)	FAO
3Mtit	OctM(P)	F	FAO
4c	OctM(P)c	F	FAO
4NADH	OctM(P)cNADH	F	FAO
5P	PMOct(P)	NF	CI&FAO
6G	PGMOct(P)	NF	CI&FAO
7S	PGMSOct(P)	NFS	CI&II&FAO
8Gp	PGMSOctGp(P)	NFSGp	CI&II&FAO&GpDH
9U	PGMSOctGp(E)	NFSGp	CI&II&FAO&GpDH
10Rot	SGp(E)	SGp	CII&GpDH
11Ama		ROX	ROX
12Tm	Tm(E)	Tm	CIV
13Azd		ROX	ROX

Laboratory sheets for SUIT protocols RP1 and RP2

Type	SUIT protocol	Sample	Abbr	docx file	pdf file	Last update
Titration sheet	RP1	Permeabilized cells	pce	»SUIT_RP1pce	»SUIT_RP1pce	2016-07-08
Titration sheet	RP2		pce	»SUIT_RP2pce	»SUIT_RP2pce	2016-07-07
Tick boxes	Combined		pce	»RP-pce print	»RP-pce print
Titration sheet	RP1	Isolated mitochondria, tissue homogenate, permeabilized fibers	imt, thom, pf	»SUIT_RP1mt	»SUIT_RP1mt	2016-07-08
Titration sheet	RP2		imt, thom, pf	»SUIT_RP2mt	»SUIT_RP2mt	2016-07-07
Tick boxes	Combined		imt, thom, pf	»RP print	»RP print

DatLab-Excel templates for RP analysis

» »DL-Excel RP for mt-preparations

» »DL-Excel RP for permeabilized cells

Experimental details

Work in progress towards the next pre-publication version of »MiPNet21.06 SUIT reference protocol.pdf«

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.

RP-T01

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

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. - Gnaiger Erich 03:23, 18 January 2016 (CET)

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 PBMCs and platelets. - Gnaiger Erich 18:47, 19 January 2016 (CET)