Difference between revisions of "MiPNet21.06 SUIT RP"
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---- | == SUIT protocol-RP1 == | ||
[[File:SUIT-Reference-Protocol 2.png|700px |SUIT RP2]]<br /> | |||
[[File:SUIT-Reference-Protocol 1.png|right|700px|link=SUIT-RP1 |SUIT-RP1]]<br /> | |||
:::: '''SUIT states:''' 1-4[[PM]](LPcE) 5[[PGM]] 6[[PGMS]] 7[[PGMSOct]] 8[[S]] 9[[SGp]] 10[[ROX]] 11[[Tm]] 12[[ROX]] | |||
{| class="wikitable" border="1" | |||
|- | |||
! 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 == | |||
[[File:SUIT-Reference-Protocol 2.png|right|700px|link=SUIT-RP2 |SUIT-RP2]]<br /> | |||
'''States:''' 1[[ROX]] 2[[Oct]](P) 3[[MOct]](4c) 5[[PMOct]] 6[[PGMOct]] 7[[PGMSOct]] 8[[PGMSOctGp]](9E) 10[[SGp]] 11[[ROX]] 12[[CIV]] 13[[ROX]] | |||
{| class="wikitable" border="1" | |||
|- | |||
! 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 | |||
|} | |||
---- | ---- | ||
{| class="wikitable" border="1" | {| class="wikitable" border="1" |
Revision as of 12:01, 20 July 2016
SUIT reference protocol for OXPHOS analysis by high-resolution respirometry. |
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
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
SUIT protocol-RP1
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
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 |
Type | SUIT protocol | Sample | Abbr | docx file | pdf file | Last update |
---|---|---|---|---|---|---|
Titration sheet | RP1 | Permeabilized cells | Pc | »SUIT_RP1Pc | »SUIT_RP1Pc | 2016-07-08 |
Titration sheet | RP2 | Pc | »SUIT_RP2Pc | »SUIT_RP2Pc | 2016-07-07 | |
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 | 2016-07-08 |
Titration sheet | RP2 | Imt, Thom, Pfi | »SUIT_RP2mt | »SUIT_RP2mt | 2016-07-07 | |
Tick boxes | Combined | Imt, Thom, Pfi | »RP print | »RP print |
DatLab-Excel templates for RP analysis
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