MiPNet21.06 SUIT RP: Difference between revisions
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{{Publication | {{Publication | ||
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] SUIT reference protocol for OXPHOS analysis by high-resolution respirometry. | |title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] SUIT reference protocol for OXPHOS analysis by high-resolution respirometry. | ||
|info=[[Media:MiPNet21.06 SUIT reference protocol.pdf |»Bioblast pdf«]] | |||
|authors=OROBOROS | |authors=OROBOROS | ||
|year=2016-01-23 | |year=2016-01-23 | ||
|journal=Mitochondr Physiol Network | |journal=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.''' | |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.''' » [http://www.bioblast.at/index.php/File:MiPNet21.06_SUIT_reference_protocol.pdf Versions] | ||
:» MitoPedia: [[SUIT reference protocol]] | :» MitoPedia: [[SUIT reference protocol]] | ||
:» Instrument: [[Oxygraph-2k|O2k]], [[OROBOROS O2k-Catalogue | O2k-Catalogue]] | :» Instrument: [[Oxygraph-2k|O2k]], [[OROBOROS O2k-Catalogue | O2k-Catalogue]] | ||
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== RP == | == RP == | ||
* RP1: Compared to RP1-T01, move Oct titration after S. | ::::* RP1: Compared to RP1-T01, move Oct titration after S. | ||
* RP2: Compared to RP2-T01, move U titration after Gp. | ::::* RP2: Compared to RP2-T01, move U titration after Gp. | ||
== RP-T01 == | == RP-T01 == | ||
=== Cytochrome c test === | === 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. | ::::* 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. | ||
:~ [[User:Gnaiger Erich|Gnaiger Erich]] 18:26, 23 January 2016 (CET) | ::::~ [[User:Gnaiger Erich|Gnaiger Erich]] 18:26, 23 January 2016 (CET) | ||
=== Mark names === | === 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. | ::::* 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. | ||
:~ [[User:Gnaiger Erich|Gnaiger Erich]] 16:50, 23 January 2016 (CET) | ::::~ [[User:Gnaiger Erich|Gnaiger Erich]] 16:50, 23 January 2016 (CET) | ||
Line 132: | Line 132: | ||
=== Saturating ADP concentrations === | === 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. | :::: 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 == | == 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 [[Gentle_Science#The_Vancouver_Protocol.2C_ICMJE.2C_and_Gentle_Science |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. - [[User:Gnaiger Erich|Gnaiger Erich]] 03:23, 18 January 2016 (CET) | :::: This communication is a pre-publication, inviting critical feedback and comments. All feedback will be carefully documented and evaluated in terms of justification of [[Gentle_Science#The_Vancouver_Protocol.2C_ICMJE.2C_and_Gentle_Science |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. - [[User:Gnaiger Erich|Gnaiger Erich]] 03:23, 18 January 2016 (CET) | ||
=== Experiments in progress === | === 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. - [[User:Gnaiger Erich|Gnaiger Erich]] 18:47, 19 January 2016 (CET) | :::: 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. - [[User:Gnaiger Erich|Gnaiger Erich]] 18:47, 19 January 2016 (CET) | ||
[[Image:MitoFit.jpg|left|60px|link=http://www.mitofit.org/index.php/K-Regio MitoFit|K-Regio MitoFit]] | [[Image:MitoFit.jpg|left|60px|link=http://www.mitofit.org/index.php/K-Regio MitoFit|K-Regio MitoFit]] | ||
* [[AT Innsbruck Gnaiger E]] and [[AT Innsbruck OROBOROS]] - [[K-Regio MitoFit |MitoFit]] project. | ::::* [[AT Innsbruck Gnaiger E]] and [[AT Innsbruck OROBOROS]] - [[K-Regio MitoFit |MitoFit]] project. | ||
* [[US NC Winston-Salem Molina AJA]] - UPBEAT project. | ::::* [[US NC Winston-Salem Molina AJA]] - UPBEAT project. | ||
== Further details == | == Further details == | ||
: | ::::» Introduction: [[Gnaiger 2014 MitoPathways]] | ||
:» [[MitoPedia: SUIT protocols]] - '''''extended MitoPedia topic, Jan 2016''''' | ::::» [[MitoPedia: SUIT protocols]] - '''''extended MitoPedia topic, Jan 2016''''' | ||
:» [[MitoPedia: SUIT states]] - '''''extended MitoPedia topic, Jan 2016''''' | ::::» [[MitoPedia: SUIT states]] - '''''extended MitoPedia topic, Jan 2016''''' | ||
:* Table of titrations » [[MiPNet09.12 O2k-Titrations]] | ::::* Table of titrations » [[MiPNet09.12 O2k-Titrations]] | ||
: | ::::» Definition: [[Substrate-uncoupler-inhibitor titration]] | ||
: | ::::» Context: [[SUIT protocol library]] | ||
: | ::::» Abbreviations: [[MitoPedia]] |
Revision as of 13:03, 2 March 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, 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
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
Type | SUIT protocol | Sample | Abbr | docx file | pdf file | Last update |
---|---|---|---|---|---|---|
Titration sheet | RP1 | Permeabilized fibres | Pfi | »SUIT_RP1-Pfi | »SUIT_RP1-Pfi | 2016-02-11 |
Titration sheet | RP2 | Pfi | »SUIT_RP2-Pfi | »SUIT_RP2-Pfi | 2016-02-11 | |
Tick boxes | Combined | Pfi | »RP print-Pfi | »RP print-Pfi | 2016-01-30 | |
Titration sheet | RP1 | Permeabilized cells | Pc | »SUIT_RP1-Pc | »SUIT_RP1-Pc | 2016-01-25 |
Titration sheet | RP2 | Pc | »SUIT_RP2-Pc | »SUIT_RP2-Pc | 2016-01-25 | |
Tick boxes | Combined | Pc | »RP-Pc print | »RP-Pc print | ||
Titration sheet | RP1 | Isolated mitochondria, tissue homogenate | Imt, Thom | »SUIT_RP1-Imt | »SUIT_RP1-Imt | 2016-01-25 |
Titration sheet | RP2 | Imt, Thom | »SUIT_RP2-Imt | »SUIT_RP2-Imt | 2016-01-25 | |
Tick boxes | Combined | Imt, Thom | »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 protocols - extended MitoPedia topic, Jan 2016
- » MitoPedia: SUIT states - extended MitoPedia topic, Jan 2016
- Table of titrations » MiPNet09.12 O2k-Titrations
- » Definition: Substrate-uncoupler-inhibitor titration
- » Context: SUIT protocol library
- » Abbreviations: MitoPedia