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, Gnaiger (2016) SUIT reference protocol for OXPHOS analysis by high-resolution respirometry. Mitochondr Physiol Network 21.06(pre03):1-10. » 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 

RP

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

RP-T01

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)

Laboratory sheets for SUIT protocols RP1 and RP2

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Type	SUIT protocol	Sample	Abbr	docx file	pdf file	Last update
Titration sheet	RP1	Permeabilized cells	Pc	»SUIT_RP1Pc	»SUIT_RP1Pc	2016-07-01
Titration sheet	RP2		Pc	»SUIT_RP2Pc	»SUIT_RP2Pc	2016-07-01
Tick boxes	Combined		Pc	»RP-Pc print	»RP-Pc print
Titration sheet	RP1	Isolated mitochondria, tissue homogenate, permeabilized fibers	Imt, Thom, Pfi	»SUIT_RP1mt	»SUIT_RP1mt	2016-07-01
Titration sheet	RP2		Imt, Thom, Pfi	»SUIT_RP2mt	»SUIT_RP2mt	2016-07-01
Tick boxes	Combined		Imt, Thom, Pfi	»RP print	»RP print

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.

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)

• AT Innsbruck Gnaiger E and AT Innsbruck OROBOROS - MitoFit project.
• US NC Winston-Salem Molina AJA - UPBEAT project.

Further details

» Introduction: Gnaiger 2014 MitoPathways

» MitoPedia: SUIT - extended MitoPedia topic, May 2016

• Table of titrations » MiPNet09.12 O2k-Titrations

» Definition: Substrate-uncoupler-inhibitor titration

» Context: SUIT protocol library

» Abbreviations: MitoPedia